Proteopedia - User contributions [en]

Variola Topoisomerase 1B

2013-11-26T20:44:44Z

Krista Cowan: New page: <StructureSection load='3igc' size='350' side='right' caption='Structure of Variola Topoisomerase 1B with DNA (PDB entry 3igc)' scene=''> [[Image:smallpox-structure-500.jpg|left|180px...

<StructureSection load='3igc' size='350' side='right' caption='Structure of Variola Topoisomerase 1B with DNA (PDB entry [[3igc]])' scene=''>
[[Image:smallpox-structure-500.jpg|left|180px]]<ref name="pic">[http://www.rkm3d.com/VIRUS/SMALLPOX/smallpox-structure-500.jpg]</ref>

===General Functions of the Protein===
Variola Topoisomerase is a Type 1B topoisomerase present in the poxviruses, the causative agent of smallpox<ref name='Perry 1'>PMID:16885024</ref>. As with most DNA encoded viruses, the poxvirus utilizes Type IB topoisomerase to relieve supercoils during processes such as replication, transcription, and repair<ref name='Textbook'>Berg, Jeremy, Yymoczko, John, Stryer, Lubert. Biochemistry 6th Edition. New York: W.H. Freeman and Company, 2007. Textbook. ISBN=9780716787242</ref>. To accomplish this relief, the enzyme ''nicks'' a single strand of the DNA molecule of interest, allowing rotation around the uncleaved strand<ref name='Stayley'>PMID:20187656</ref>. This action removes between 5 to 14 superhelical turns each time the DNA molecule is nicked<ref name='Stayley' />. In order to better understand the mechanism of Variola Topoisomerase's major function, we will first look at the structural details.

===Structural Details===
Variola Topoisomerase is one of the smallest known topoisomerases, measuring a mere 34 kDA<ref name='Perry 2'>PMID:20152159</ref>. The enzyme is folded into two domains: <scene name='56/565703/Domains/1'>the N domain and the catalytic domain</scene><ref name='Perry 1' />. The N domain consists of a 5 stranded antiparallel beta sheet (labeled β1-5) and 2 alpha helixes (labeled α1-2) twisted together<ref name='Perry 1' />. The catalytic domain consists of a 3 stranded beta sheet (labeled β6-8) and alpha helixes(labeled α4-12)<ref name='Perry 1' />. The catalytic domain sits on the minor groove face while the active is positioned on the major groove face of the DNA sequence. The two domains are bound by the core sequence 5'-CCCTT-3'<ref name='Stayley' />. This binding creates the observable C-shaped clamp surrounding the DNA molecule being acted upon<ref name='Stayley' />. The non-covalently bound complex has a salt bridge not observed in the covalently bound complex. The <scene name='56/565703/Salt_bridge/2'>salt bridge</scene> connects the Lys65 found within the N domain to the Glu139 of the catalytic domain<ref name='Perry 1' />. The enzyme can either be covalently or non-covalently bonded to the DNA molecule only differing at the active site.

===Active Site===
The key residue for the differences observed between the covalently and non-covalently bound states is Tyrosine 274<ref name='Perry 1' />. When cleaving the single strand of DNA, Tyr274 acts as a nucleophile attacking the hydroxyl group of the O'5<ref name='Perry 1' />. When ligating the strand back together, the roles reverse, allowing the 5' hydroxyl group to nucleophilicly attack the Tyr274 residue<ref name='Perry 1' />. Amazingly, the enzyme is able to alter between these two bound states without utilizing any high energy cofactors, though there are several other significant residues within the active site that promote this mechanism, namely <scene name='56/565703/Transition_state_active_site_2/1'>Arg130, Arg223, His265, and Lys167</scene><ref name='Perry 1' />. Three residues of the active site serve to stabilize the buildup of negative charges in the transition state as Tyr274 nucleophilically attacks: Arg130, Arg223, and His265<ref name='Perry 1' />. This is accomplished by the residues forming hydrogen bonds to the scissle phosphate<ref name='Perry 1' />. Lys167 makes direct contact with the DNA molecule at the +1 base of the scissle phosphate<ref name='Perry 1' />. In order to make the O5'-hydroxyl group a better leaving group, the Arg130 and Lys167 residues contribute to the protonation of the hydroxyl group<ref name='Perry 1' />. Currently, a water molecule found within the active site is theorized to act as the acid/base catalyst for Tyr274 hydroxyl group<ref name='Perry 2' />. In the non-covalently bound complex, the measured distance of the <scene name='56/565703/Non-covalently_bound_tyr274/4'>Tyr274 hydroxyl group relative to the scissle phosphate</scene> is approximately 8Å<ref name='Perry 1' />. Thus, during the conformation change, i.e. the movement of the α10a-α10b segment, results in the movement of the Tyr274 residue by approximately 3.6Å<ref name='Perry 2' />.

===Downstream Interactions===
In addition to the active site, Variola Topoisomerase 1B has downstream contacts that assist with its binding to the DNA. There are four different interaction points of Topoisomerase that interact for this purpose: the N-terminal domain, the conserved β turn, the α9 helix, and the α10a helix<ref name='Perry 2' />.

First, Lys35 and His39 side chains on the N-terminal domain form hydrogen bonds and van der Waals interaction with the uncleaved DNA strand<ref name='Perry 2' />.

In the conserved β turn, adjacent to the cleavage site, Asp168 makes contact with the DNA at the -1 sugar<ref name='Perry 2' />. The <scene name='56/565703/Transition_state_asp168/1'>Asp side chain</scene> forms a water-mediated hydrogen bond with N3 of +A on the uncleaved strand<ref name='Perry 2' />. During the transition state, it also interacts with sugars on the opposite face of the minor groove<ref name='Perry 2' />. This includes contact with carboxylate oxygen and carbon atoms of the -1A sugar, the leaving group during the cleavage of DNA, and the +1 sugar of the uncleaved strand<ref name='Perry 2' />. The CH-O bonds made by Asp168 influences ring position and allow Lys167 to affect the torsion angle of the C4’-C5’ bond<ref name='Perry 2' />. Through these interactions, Asp168 serves to promote ligation, by catching the rotating downstream DNA and positioning it for ligation<ref name='Perry 2' />.

The rest of the downstream interactions occur in the α9 and α10a helices of Topoisomerase 1B<ref name='Perry 2' />. The α10a helix is in the major groove and interacts with the nucleotides -3 to -1 on the cleaved strand to influence the rate of DNA cleavage<ref name='Perry 2' />. Three main amino acids assist with the interaction: <scene name='56/565703/Transition_state_alpha_10/5'>Thr266, Ile269, and Arg272</scene>. Thr266’s amide nitrogen and hydroxyl group hydrogen bond to the -1/-2 phosphate<ref name='Perry 2' />. Meanwhile, Arg272 bridges the O4 groups of the -2T and -3T bases with hydrogen bonds<ref name='Perry 2' />. Ille269 facilitates van der Waals interactions with the -2T base and the -1A sugar<ref name='Perry 2' />. In addition to these three, Lys271 interacts with the -5/-6 phosphate on the noncleaved strand<ref name='Perry 2' />. All of these interactions are essential because the α10a also includes Tyr274 at its C-terminus<ref name='Perry 2' />. These interactions allow the correct positioning of this essential catalytic residue.

The last important downstream contact site is approximately 10bp downstream of the catalytic site at the α9 helix<ref name='Perry 2' />. In this helix, Lys250 and Lys249 hydrogen bond to the -10/-11 phosphate and Lys249 to the -6/-7 phosphate of the uncleaved strand<ref name='Perry 2' />. Additionally, Ala253 forms hydrogen bonds with the sugar-phosphate backbone of the uncleaved strand, pulling it within a distance of 4.3Å<ref name='Perry 2' />. These interactions allow the DNA to come in close proximity to the Topoisomerase 1B.

===Specificity===
What gives Variola Topoisomerase its specificity is that it can only cleave and ligate DNA at a pentapryimidine sequence 5'-(T/C)CCTT-3'<ref name='Stayley' />. More specifically, this is a phosphate group found after the 3'-terminal thymidine<ref name='Stayley' />. Cleavage at this specific sequence is a nearly impossible feat for human strains of Type I Topoisomerases<ref name='Stayley' />. The beta 5 strand of the N domain makes 3 direct contacts with the major groove face of the DNA molecule at positions Tyr70, Tyr72, and Gln69<ref name='Perry 1' />. Tyr70 hydrogen bonds to the phosphate backbone while also interacting with the Cyt+3 and Cyt+4 bases<ref name='Perry 1' />. Tyr72 also hydrogen bonds to the phosphate backbone while interacting with the +3 ribose ring and the Thy+2 base<ref name='Perry 1' />. Gln69 makes a double hydrogen bond with Ade+2<ref name='Stayley' />. The variola strain also shows a talent for discriminating against DNA strains with mismatches, giving greatest significance to those at the 1', 2', or 3' positions relative to the cleavage site<ref name='Stayley' />. Studies have shown that the equilibrium constants and reversible strand ligation forward rate will decrease 10 times when these mismatches are present<ref name='Stayley' />.

===See Also===
----
*[[3igc|3icg]]
*[[2h7f|2h7f]]
*[[2h7g|2h7g]]

===References:===
----

{{reflist}}

</StructureSection>

Sandbox krista

2013-10-29T15:39:23Z

Krista Cowan: New page: == Your Heading Here (maybe something like 'Structure') == <StructureSection load='1qln' size='350' side='right' caption='Structure of HMG-CoA reductase (PDB entry 1qln)' scene=''> Any...

== Your Heading Here (maybe something like 'Structure') ==
<StructureSection load='1qln' size='350' side='right' caption='Structure of HMG-CoA reductase (PDB entry [[1qln]])' scene=''>
Anything in this section will appear adjacent to the 3D structure and will be scrollable.

</StructureSection>

Ann Taylor Sandbox 4

2013-10-07T18:41:24Z

Krista Cowan:

{{STRUCTURE_2ada| PDB=2ada | SCENE= }}

===ADENOSINE DEAMINASE===
by Krista Cowan

Adenosine deaminase is involved in the degradation of purine nucleotides. It is especially active in lympocytes, and mutation of adenosine deaminase results in severe immunodeficiency. Adenosine deaminase contains an <scene name='36/365339/Beta_barrel/1'>eight stranded parallel alpha/beta barrel</scene> with the active site in a deep pocket at the beta-barrel COOH-terminal end. <ref>PMID:1925539 </ref> The active site contains a <scene name='36/365339/Zinc_co-factor/1'>Zinc cofactor</scene>, which coordinates to the 6-hydroxyl of the transition state analogue, 6-hydroxyl, 1,6-dihydropurine ribonucleoside. The zinc is coordinated to
<scene name='36/365339/Histines_and_asp_residues/1'>three histidine residues and an aspartic acid residue</scene>.

The transition state analogue held in place mostly by polar interactions. The ribose group is close to the opening of the pocket, with the purine portion deeper in the pocket, close to the zinc. Nine hydrogen bonds stabilize the transition state-enzyme complex.

ADA is very stereoselective for the 6R isomer. This specificity is due to the location of the catalytic zinc, Asp295 and His 238. Interestingly, one face of the purine ring is exposed to polar groups and zinc, while the other face is only exposed to nonpolar residues. The proposed catalytic mechanism has Asp295 act as a general base, while the zinc acts as an electrophile to activate the water molecule. His 238 orients the water and stabilizes the charge of the attacking hydroxide. The protonated <scene name='36/365339/Glu217/1'>Glu217</scene> or the water hydrogen bonded to it could donate or share a proton with the N1 of the substrate.

<references/>

Ann Taylor Sandbox 4

2013-10-07T18:20:27Z

Krista Cowan:

{{STRUCTURE_2ada| PDB=2ada | SCENE= }}

===ADENOSINE DEAMINASE===
by Krista Cowan

Adenosine deaminase is involved in the degradation of purine nucleotides. It is especially active in lympocytes, and mutation of adenosine deaminase results in severe immunodeficiency. Adenosine deaminase contains an <scene name='36/365339/Beta_barrel/1'>eight stranded parallel alpha/beta barrel</scene> with the active site in a deep pocket at the beta-barrel COOH-terminal end. <ref>PMID:1925539 </ref> The active site contains a <scene name='36/365339/Zinc_co-factor/1'>Zinc cofactor</scene>, which coordinates to the 6-hydroxyl of the transition state analogue, 6-hydroxyl, 1,6-dihydropurine ribonucleoside. The zinc is coordinated to
<scene name='36/365339/Histines_and_asp_residues/1'>three histidine residues and an aspartic acid residue</scene>.

The transition state analogue held in place mostly by polar interactions. The ribose group is close to the opening of the pocket, with the purine portion deeper in the pocket, close to the zinc. Nine hydrogen bonds stabilize the transition state-enzyme complex.

ADA is very stereoselective for the 6R isomer. This specificity is due to the location of the catalytic zinc, Asp295 and His 238. Interestingly, one face of the purine ring is exposed to polar groups and zinc, while the other face is only exposed to nonpolar residues. The proposed catalytic mechanism has Asp295 act as a general base, while the zinc acts as an electrophile to activate the water molecule. His 238 orients the water and stabilizes the charge of the attacking hydroxide. The protonated Glu217 or the water hydrogen bonded to it could donate or share a proton with the N1 of the substrate.

<references/>

Ann Taylor Sandbox 4

2013-10-07T17:53:21Z

Krista Cowan:

{{STRUCTURE_2ada| PDB=2ada | SCENE= }}

===ADENOSINE DEAMINASE===
by Krista Cowan

Adenosine deaminase is involved in the degradation of purine nucleotides. It is especially active in lympocytes, and mutation of adenosine deaminase results in severe immunodeficiency. Adenosine deaminase contains an <scene name='36/365339/Beta_barrel/1'>eight stranded parallel alpha/beta barrel</scene> with the active site in a deep pocket at the beta-barrel COOH-terminal end. <ref>PMID:1925539 </ref> The active site contains a <scene name='36/365339/Zinc_co-factor/1'>Zinc cofactor</scene>, which coordinates to the 6-hydroxyl of the transition state analogue, 6-hydroxyl, 1,6-dihydropurine ribonucleoside. The zinc is coordinated to three histidine residues and an aspartic acid residue.

The transition state analogue held in place mostly by polar interactions. The ribose group is close to the opening of the pocket, with the purine portion deeper in the pocket, close to the zinc. Nine hydrogen bonds stabilize the transition state-enzyme complex.

ADA is very stereoselective for the 6R isomer. This specificity is due to the location of the catalytic zinc, Asp295 and His 238. Interestingly, one face of the purine ring is exposed to polar groups and zinc, while the other face is only exposed to nonpolar residues. The proposed catalytic mechanism has Asp295 act as a general base, while the zinc acts as an electrophile to activate the water molecule. His 238 orients the water and stabilizes the charge of the attacking hydroxide. The protonated Glu217 or the water hydrogen bonded to it could donate or share a proton with the N1 of the substrate.

<references/>

Ann Taylor Sandbox 4

2013-10-07T17:42:23Z

Krista Cowan:

{{STRUCTURE_2ada| PDB=2ada | SCENE= }}

===ADENOSINE DEAMINASE===
by Krista Cowan

Adenosine deaminase is involved in the degradation of purine nucleotides. It is especially active in lympocytes, and mutation of adenosine deaminase results in severe immunodeficiency. Adenosine deaminase contains an eight stranded parallel alpha/beta barrel with the active site in a deep pocket at the beta-barrel COOH-terminal end. <ref>PMID:1925539 </ref> The active site contains a <scene name='36/365339/Zinc_co-factor/1'>Zinc cofactor</scene>, which coordinates to the 6-hydroxyl of the transition state analogue, 6-hydroxyl, 1,6-dihydropurine ribonucleoside. The zinc is coordinated to three histidine residues and an aspartic acid residue.

The transition state analogue held in place mostly by polar interactions. The ribose group is close to the opening of the pocket, with the purine portion deeper in the pocket, close to the zinc. Nine hydrogen bonds stabilize the transition state-enzyme complex.

ADA is very stereoselective for the 6R isomer. This specificity is due to the location of the catalytic zinc, Asp295 and His 238. Interestingly, one face of the purine ring is exposed to polar groups and zinc, while the other face is only exposed to nonpolar residues. The proposed catalytic mechanism has Asp295 act as a general base, while the zinc acts as an electrophile to activate the water molecule. His 238 orients the water and stabilizes the charge of the attacking hydroxide. The protonated Glu217 or the water hydrogen bonded to it could donate or share a proton with the N1 of the substrate.

<references/>

Ann Taylor Sandbox 4

2013-10-07T16:39:38Z

Krista Cowan:

{{STRUCTURE_2ada| PDB=2ada | SCENE= }}

===ADENOSINE DEAMINASE===
by Krista Cowan

Adenosine deaminase is involved in the degradation of purine nucleotides. It is especially active in lympocytes, and mutation of adenosine deaminase results in severe immunodeficiency. Adenosine deaminase contains an eight stranded parallel alpha/beta barrel with the active site in a deep pocket at the beta-barrel COOH-terminal end. <ref>PMID:1925539 </ref> The active site contains a zinc cofactor, which coordinates to the 6-hydroxyl of the transition state analogue, 6-hydroxyl, 1,6-dihydropurine ribonucleoside. The zinc is coordinated to three histidine residues and an aspartic acid residue.

The transition state analogue held in place mostly by polar interactions. The ribose group is close to the opening of the pocket, with the purine portion deeper in the pocket, close to the zinc. Nine hydrogen bonds stabilize the transition state-enzyme complex.

ADA is very stereoselective for the 6R isomer. This specificity is due to the location of the catalytic zinc, Asp295 and His 238. Interestingly, one face of the purine ring is exposed to polar groups and zinc, while the other face is only exposed to nonpolar residues. The proposed catalytic mechanism has Asp295 act as a general base, while the zinc acts as an electrophile to activate the water molecule. His 238 orients the water and stabilizes the charge of the attacking hydroxide. The protonated Glu217 or the water hydrogen bonded to it could donate or share a proton with the N1 of the substrate.

<references/>

User:Krista Cowan

2013-10-03T03:36:22Z

Krista Cowan:

Krista Cowan
Biological Sciences: Biochemistry and Cellular and Molecular Biology Major
I am a student in BCMB 402 at the University of Tennessee in Knoxville, Tennessee in the United States.

Ann Taylor Sandbox 4

2013-10-03T03:32:36Z

Krista Cowan:

{{STRUCTURE_2ada| PDB=2ada | SCENE= }}

===ADENOSINE DEAMINASE===

Adenosine deaminase is involved in the degradation of purine nucleotides. It is especially active in lympocytes, and mutation of adenosine deaminase results in severe immunodeficiency. Adenosine deaminase contains an eight stranded parallel alpha/beta barrel with the active site in a deep pocket at the beta-barrel COOH-terminal end. <ref>PMID:1925539 </ref> The active site contains a zinc cofactor, which coordinates to the 6-hydroxyl of the transition state analogue, 6-hydroxyl, 1,6-dihydropurine ribonucleoside. The zinc is coordinated to three histidine residues and an aspartic acid residue.

The transition state analogue held in place mostly by polar interactions. The ribose group is close to the opening of the pocket, with the purine portion deeper in the pocket, close to the zinc. Nine hydrogen bonds stabilize the transition state-enzyme complex.

ADA is very stereoselective for the 6R isomer. This specificity is due to the location of the catalytic zinc, Asp295 and His 238. Interestingly, one face of the purine ring is exposed to polar groups and zinc, while the other face is only exposed to nonpolar residues. The proposed catalytic mechanism has Asp295 act as a general base, while the zinc acts as an electrophile to activate the water molecule. His 238 orients the water and stabilizes the charge of the attacking hydroxide. The protonated Glu217 or the water hydrogen bonded to it could donate or share a proton with the N1 of the substrate.

<references/>