Sandbox Reserved 1068: Difference between revisions
From Proteopedia
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 22: | Line 22: | ||
== Structural highlights == | == Structural highlights == | ||
[[Image:Screen Shot 2015-04-26 at 6.31.19 PM.png|200 px|left|thumb|'''Figure 3:''' Overlay of chain A in [[3ST6]] (green) and [[3RV6]] (teal). [[3ST6]] contains the inhibitor AMT and represents the closed form of MbtI while [[3RV6]] contains an enolpyruvyl modified inhibitor (phenyl-AMT) and shows the movement of the backbone away from the closed form to accommodate the modified inhibitor. <ref name= "8a"/>.]]. | |||
[[Image:Screen Shot 2015-04-26 at 6.29.56 PM.png|200 px|left|thumb|'''Figure 4:''' Overlay of chain A in [[3ST6]] (green) and [[3RV6]] (teal) with inhibitors bound and magnesium. <ref name= "8a"/>.]] | |||
[[Image:Screen Shot 2015-04-26 at 6.30.55 PM.png|200 px|left|thumb|'''Figure 5:''' Flexibility of peptide backbone in regions proximal to the active site see through the overlay of chain A residues 269-293 and 324-336 in [[3ST6]] (green) and [[3RV6]] (teal)<ref name= "8a"/>.]]. | |||
MbtI structure has a mobile element (residues 268-293 and 324-336) that can adopt a <scene name='69/694235/Irp9_closed_state/2'>closed</scene> or <scene name='69/694235/2g5f_with_open_loop/1'>open conformation</scene> depending on whether or not ligands are bound to the active site.<ref name="3a"/> The closed conformation partially obstructs the active site. <ref name= "5a"/>. | MbtI structure has a mobile element (residues 268-293 and 324-336) that can adopt a <scene name='69/694235/Irp9_closed_state/2'>closed</scene> or <scene name='69/694235/2g5f_with_open_loop/1'>open conformation</scene> depending on whether or not ligands are bound to the active site.<ref name="3a"/> The closed conformation partially obstructs the active site. <ref name= "5a"/>. | ||
Inhibition studies have also shown a switch in binding mode at the MbtI active site for inhibitors carrying a substituted enolpyruvyl group compared to the chorismate substrate <ref name="5a">PMID:22607697</ref> <ref name= | Inhibition studies have also shown a switch in binding mode at the MbtI active site for inhibitors carrying a substituted enolpyruvyl group compared to the chorismate substrate <ref name="5a">PMID:22607697</ref> <ref name= | ||
"2a">PMID:23108268</ref> <ref name="1a">PMID:20512795</ref>. Crystal structures and fluorescent-based thermal shift assays show that substituents larger than a methyl group are accommodated in the active site of MbtI through localized flexibility in the peptide backbone.<ref name="5a"/> Positioning of the <scene name='69/694235/3st6_structure_bindingpocket/3'>active site residues</scene> of MbtI in [[3ST6]] with the inhibitor AMT bound is highly similar to the positioning of the <scene name='69/694235/3log_bindingpocket/3'>active site residues</scene> in closed form of MbtI [[3log]] with succinic acid bound <ref name= "5a"/>. The AMT inhibitor contains an unmodified enolpyruvyl side chain and resembles the structure of the natural substrate, chorismate. [[3log]] and [[3ST6]] are shown to share a similar binding mode, termed binding mode 1. Isochorismate inhibitors with modified enolpyruvl side chains ([[3VEH]], [[3RV9]], [[3RV8]], [[3RV7]], [[3RV6]]) utilize a novel binding mode, termed mode 2, which involves the <scene name='69/694235/3veh_structure_bindingpocket2/2'>reorientation of the isochorismate analogue within the active site</scene>. Movement of the peptide backbone away from the closed form of MbtI is required to accommodate the enolpyruyl modified inhibitors. | "2a">PMID:23108268</ref> <ref name="1a">PMID:20512795</ref>. Crystal structures and fluorescent-based thermal shift assays show that substituents larger than a methyl group are accommodated in the active site of MbtI through localized flexibility in the peptide backbone.<ref name="5a"/> Positioning of the <scene name='69/694235/3st6_structure_bindingpocket/3'>active site residues</scene> of MbtI in [[3ST6]] with the inhibitor AMT bound is highly similar to the positioning of the <scene name='69/694235/3log_bindingpocket/3'>active site residues</scene> in closed form of MbtI [[3log]] with succinic acid bound <ref name= "5a"/>. The AMT inhibitor contains an unmodified enolpyruvyl side chain and resembles the structure of the natural substrate, chorismate. [[3log]] and [[3ST6]] are shown to share a similar binding mode, termed binding mode 1. Isochorismate inhibitors with modified enolpyruvl side chains ([[3VEH]], [[3RV9]], [[3RV8]], [[3RV7]], [[3RV6]]) utilize a novel binding mode, termed mode 2, which involves the <scene name='69/694235/3veh_structure_bindingpocket2/2'>reorientation of the isochorismate analogue within the active site</scene>. Movement of the peptide backbone away from the closed form of MbtI is required to accommodate the enolpyruyl modified inhibitors. | ||
==Molecular Mechanism== | ==Molecular Mechanism== | ||
'''Magnesium cation effect''' | '''Magnesium cation effect''' | ||
[[Image:Screen Shot 2015-04-25 at 11.08.58 PM.png |300 px|left|thumb|'''Table 1:''' pKa values of active site residues of MbtI with and without Magnesium. <ref name="8a"/> 2-11.]] The presence of the [http://en.wikipedia.org/wiki/Magnesium_in_biology magnesium ion] induces changes in the structure of the active site and in the substrate, as well as causes significant pKa shifts in some of the key residues involved in the catalytic activity (Table 1).The <scene name='69/694235/3rv6_mg_shell/4'>coordination shell</scene>of the magnesium cation in the active site of MbtI in [[3rv6]] with phenyl-AMT inhibitor bound is composed of two water molecules, Glu434, Glu294, and the two oxygen atoms of the C1 carboxylate group of chorismate. In the presence of the magnesium ion, the positively charged Lys295 is displaced from the active site and the negatively charged Glu297 is faced toward the active site. Magnesium cation also orients the C1 carboxylate group coplanar to the ring of chorismate, reducing the electron density on the C2 center and favoring nucleophilic attack<ref name= "8a">PMID:22307014</ref>. | [[Image:Screen Shot 2015-04-25 at 11.08.58 PM.png |300 px|left|thumb|'''Table 1:''' pKa values of active site residues of MbtI with and without Magnesium. <ref name="8a"/> 2-11.]] The presence of the [http://en.wikipedia.org/wiki/Magnesium_in_biology magnesium ion] induces changes in the structure of the active site and in the substrate, as well as causes significant pKa shifts in some of the key residues involved in the catalytic activity (Table 1).The <scene name='69/694235/3rv6_mg_shell/4'>coordination shell</scene>of the magnesium cation in the active site of MbtI in [[3rv6]] with phenyl-AMT inhibitor bound is composed of two water molecules, Glu434, Glu294, and the two oxygen atoms of the C1 carboxylate group of chorismate. In the presence of the magnesium ion, the positively charged Lys295 is displaced from the active site and the negatively charged Glu297 is faced toward the active site. Magnesium cation also orients the C1 carboxylate group coplanar to the ring of chorismate, reducing the electron density on the C2 center and favoring nucleophilic attack<ref name= "8a">PMID:22307014</ref>. | ||
