Sandbox Reserved 1068: Difference between revisions

The crystal asymmetric unit was found to contain <scene name='69/694235/3log/11'> four MbtI molecules</scene>, however crystal packing and size exclusion chromatography data suggest a monomeric enzyme <ref name= "3a">PMID 16923875</ref>. There are no significant structural changes between the four monomers excepts from the localized differences in the active site <ref name= "3a"/>. The overall molecular structure consist of a polypeptide of 450 residues that forms <scene name='69/694235/Alpha_helics/2'>one large single domain</scene> with a similar fold to other chromate-utilizing enzymes <ref name="3a"/>. The core of the protein is formed by <scene name='69/694234/Beta_sheets/1'>21 Beta sheets </scene>folded into a twisted beta-sandwich. The protein's core is then surrounded by <scene name='69/694235/Beta_sheets/4'>10 alpha helices</scene><ref name="3a"/>. The active site was identified by comparison to the product bound forms of Irp9 and TrpE and is situated in a cleft that is about 12Å in length, 10Å deep, and 7Å wide <ref name="3a"/>. One side of the groove is formed by β21, C-terminal helix, and α11 while the other side of the groove is formed by β16-17 loop, helix α7, and β15-α6 loop (figure 2)<ref name="3a"/>. The β19-20 and β12-13 loops make up the bottom of the active side cleft (Figure 2) <ref name="3a">PMID:16923875</ref>.

[[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 6.30.55 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 closed or open conformation depending on whether or not ligands are bound to the active site (Figure 3,4,5)<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="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.