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https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Girardet+Aline Proteopedia - User contributions [en] 2026-10-05T20:26:07Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340404 Sandbox Reserved 962 2015-01-09T18:12:44Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> It has been the least understood enzyme intervening in capping. To be studied, the cap methyltransferase has been isolated on purified vaccinia virus by Martin and Moss in 1975-1976.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> One of the enzymes catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. It is also necessary to keep the stability of the mRNA.<br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices A, F, H, E, I and G</scene>. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the <scene name='60/604481/Sheets_segment_1/1'>bêta-sheets from 5 to 11</scene>.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the <scene name='60/604481/Helices_segment_2/1'>alpha helices B, C, D</scene> and the <scene name='60/604481/Sheets_segment_2/1'>bêta-sheets from 1 to 4</scene>. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340403 Sandbox Reserved 962 2015-01-09T18:10:34Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> It has been the least understood enzyme intervening in capping. To be studied, the cap methyltransferase has been isolated on purified vaccinia virus by Martin and Moss in 1975-1976.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> One of the enzymes catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. It is also necessary to keep the stability of the ARN.<br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices A, F, H, E, I and G</scene>. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the <scene name='60/604481/Sheets_segment_1/1'>bêta-sheets from 5 to 11</scene>.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the <scene name='60/604481/Helices_segment_2/1'>alpha helices B, C, D</scene> and the <scene name='60/604481/Sheets_segment_2/1'>bêta-sheets from 1 to 4</scene>. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340228 Sandbox Reserved 962 2015-01-09T10:25:03Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> One of the enzymes catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices A, F, H, E, I and G</scene>. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the <scene name='60/604481/Sheets_segment_1/1'>bêta-sheets from 5 to 11</scene>.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the <scene name='60/604481/Helices_segment_2/1'>alpha helices B, C, D</scene> and the <scene name='60/604481/Sheets_segment_2/1'>bêta-sheets from 1 to 4</scene>. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340026 Sandbox Reserved 962 2015-01-08T20:56:34Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices A, F, H, E, I and G</scene>. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the <scene name='60/604481/Sheets_segment_1/1'>bêta-sheets from 5 to 11</scene>.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the <scene name='60/604481/Helices_segment_2/1'>alpha helices B, C, D</scene> and the <scene name='60/604481/Sheets_segment_2/1'>bêta-sheets from 1 to 4</scene>. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340024 Sandbox Reserved 962 2015-01-08T20:52:28Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices</scene> A, F, H, E, I and G. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the <scene name='60/604481/Sheets_segment_1/1'>bêta-sheets</scene> from 5 to 11.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the <scene name='60/604481/Helices_segment_2/1'>alpha helices</scene> B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340023 Sandbox Reserved 962 2015-01-08T20:50:13Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices</scene> A, F, H, E, I and G. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the <scene name='60/604481/Sheets_segment_1/1'>bêta-sheets</scene> from 5 to 11.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340021 Sandbox Reserved 962 2015-01-08T20:47:01Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the <scene name='60/604481/Helices_segment_1/1'>alpha helices</scene> A, F, H, E, I and G. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340013 Sandbox Reserved 962 2015-01-08T20:39:05Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the <scene name='60/604481/Helices_c_terminal/1'>C-terminal helices</scene> from the aminoacid 249 to 284. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340004 Sandbox Reserved 962 2015-01-08T20:32:08Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>first segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The <scene name='60/604481/Segment_2/1'>second segment</scene> from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2340002 Sandbox Reserved 962 2015-01-08T20:28:30Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The <scene name='60/604481/Segment_1/1'>First segment</scene> from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The second segment from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2339970 Sandbox Reserved 962 2015-01-08T19:03:01Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7) Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The first segment from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The second segment from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2339948 Sandbox Reserved 962 2015-01-08T18:15:36Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> In eukaryotic cells the synthezise of mRNA is followed by a process of maturation. The best-known modifications are the polyadenylation, the splicing and the (Guanine-N7) capping. The capping is an important step of the maturation. It consists in adding a 7-methylguanosine on the first nucleotide of the mRNA with a diphosphate bonding. <br /> The enzyme catalysing this reaction is the mRNA Cap (Guanine-N7= Methyltransferase. <br /> <br /> The cap allows the mRNA to be recognized by a protein complex which is involved in traduction initiation. <br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The first segment from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The second segment from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2335741 Sandbox Reserved 962 2015-01-07T18:29:26Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> The mRNA Cap (Guanine-N7) Methyltransferase is an enzyme which catalyses the reaction of capping the C-terminal domain of a mRNA with a m7G(5')pppR cap.<br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The first segment from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The second segment from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2335737 Sandbox Reserved 962 2015-01-07T18:23:16Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> <br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1 (the smallest cap methyltranferase identified) is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> <br /> • The first segment from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> <br /> • The second segment from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2335732 Sandbox Reserved 962 2015-01-07T18:17:37Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> <br /> <br /> '''mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)''' <br /> <br /> <br /> <br /> <Structure load='1ri1' size='350' frame='true' align='right' caption='Ecm1' scene='60/604481/Scene_depart/3' /><br /> <br /> <br /> == Biological role==<br /> <br /> == Structure ==<br /> <br /> This protein measures 292 aminoacids in lenght. <br /> <br /> The structure of ECM1, the smallest cap methyltranferase identified is similar to the structure of the whole family of cap methyltransferase. <br /> We can indentify structural repeatings characteristic of the second class of the family : amongst others alpha helices or bêta-sheets can be found. <br /> <br /> The structure of the protein could be divided in two segments : <br /> • The first segment from the aminoacids 41 to 62 and from 165 to 292 : carrys the alpha helices A, F, H, E, I and G. The helices G, H and I are the C-terminal helices from the aminoacid 249 to 2846. This segment carrys also the bêta-sheets from 5 to 11.<br /> • The second segment from the aminoacids 63 to 164 : carrys the alpha helices B, C, D and the bêta-sheets from 1 to 4. <br /> <br /> <br /> == Interaction ==<br /> <br /> === Cap Analog Binding (Guanosine 5'-Triphosphate)=== <br /> [[Image:GTP_structure.png]]<br /> <br /> <scene name='60/604481/Gtg/4'>A cap analog</scene> binds to the enzyme in a pocket near AdoHcy.{{clear}}<br /> <scene name='60/604481/Interaction_gtg/2'>6 amino acids</scene> (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine <scene name='60/604481/N1_n3_06_gtg/1'>N1, N3, and O6 atoms</scene> and with the <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2.</scene>{{clear}}<br /> The cap makes Van der Walls contacts with side chains from <scene name='60/604481/Vdw_gtg/1'>Leu216, Leu217, Asp218, Ser219.</scene> {{clear}}<br /> GTP makes a hydrogen bond with <scene name='60/604481/Hb_gtg/1'>Tyr284</scene> and a water mediated bond with <scene name='60/604481/Water_gtg/1'>Tyr145</scene>.<br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> [[Image:Inhibition_ECM1.png ]]<br /> {{clear}}<br /> As we can see on the figure above<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> , the enzyme specifically binds to guanine.{{clear}} <br /> This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the <scene name='60/604481/N1_n3_06_gtg/1'>O6 atom</scene> of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 shows that the interactions between Ecm1 and <scene name='60/604481/N2_gtg/1'>guanine exocyclic 2-NH2</scene> are important for substrate binding. {{clear}}<br /> We also remark that the methyltransferase is not able to discrminate between <scene name='60/604481/Ribose_gtg/1'>ribose</scene> and desoxyribose nucleoside sugars.<br /> <br /> <br /> <br /> === AdoHcy Binding (S-Adenosyl-L-Homocysteine)===<br /> <br /> [[Image:AdoHcy_nouveau.PNG]]<br /> <br /> The mRNA Cap Methyltransferase bind to <scene name='60/604481/Sah/2'>AdoHcy</scene> which is the product of the methyl donor AdoMet after the methylation. The interaction describe here are applicable to AdoMet too. {{clear}} <br /> AdoHcys is in a pocket formed by amino acids of segment 2.{{clear}}<br /> <scene name='60/604481/Interaction_sah/1'>10 amino acids</scene> (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.<br /> The interactions between AdoHcys and the enzyme are made of : {{clear}}<br /> - Hydrogen bonds mediated by <scene name='60/604481/Hd_sah/1'>Lys54, Gly72, Asp94, Asp122, Gln140</scene> {{clear}}<br /> - Van der Walls interactions mediated by <scene name='60/604481/Vdw_sah/1'>Ile95, Tyr124 and Ser142</scene> {{clear}}<br /> - An electrostatic interaction mediated by <scene name='60/604481/Elec_sah/1'>Gln140 and Phe141</scene> {{clear}} <br /> - A water mediated contact mediated by <scene name='60/604481/Water_sah/1'>Asp78</scene><br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> == Mechanism==<br /> This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism. {{clear}}<br /> We remark that there is no contact between the enzyme and <scene name='60/604481/S_n7/1'>the guanine N-7 nucleophile, the AdoHcy sulfur leaving group</scene> or the AdoMet methyl carbon.{{clear}}<br /> Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. <br /> <ref name="Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. ">PMID:14731396</ref><br /> <br /> ==Inhibition==<br /> <br /> [[Image:Inhibitor_structure.png|left ]] [[Image:Inhibitor.png|right]]{{clear}}<br /> <br /> *'''AdoHcy'''{{clear}}<br /> It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 is 4μm.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref> {{clear}}<br /> <br /> * '''Sinefugin'''{{clear}}<br /> Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities are probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferases.<ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref>{{clear}}<br /> <br /> *'''Aza-AdoMet & carbocyclic aza-AdoMet'''{{clear}}<br /> Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. In the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitors of Ecm1. The IC50 value of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm<br /> <ref name="Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9.">PMID:15760890 </ref><br /> <br /> ==Related Structure==<br /> <br /> [http://proteopedia.org/wiki/index.php/1ri2 1ri2] : Ecm1 associated with GTP only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri3 1ri3] : Ecm1 associated with AdoHcy only {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri4 1ri4] : Ecm1 associated with AdoMet {{clear}}<br /> [http://proteopedia.org/wiki/index.php/1ri5 1ri5] : Ecm1 only {{clear}}<br /> <br /> <br /> == References ==<br /> <references/><br /> <br /> <br /> == Proteopedia page contributors and editors ==<br /> <br /> Aline Girardet & Laure Hertzog</div>

Girardet Aline https://proteopedia.org/index.php?title=Sandbox_Reserved_962&diff=2103100 Sandbox Reserved 962 2014-12-16T12:50:53Z

<p>Girardet Aline: </p> <hr /> <div>{{Sandbox_ESBS}}<br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> <StructureSection load='1ri1' size='340' side='right' caption='[[1ri1]], [[Resolution|resolution]] 2.50&Aring;' scene=''><br /> <br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Girardet Aline