Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 46, Pages 35478-35486Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M607204200
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Funding
- MRC [G117/444] Funding Source: UKRI
- Medical Research Council [G117/444] Funding Source: researchfish
- Medical Research Council [G117/444] Funding Source: Medline
- NHLBI NIH HHS [R01 HL068629, R01 HL68629, R01 HL068629-06] Funding Source: Medline
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The poor inhibitory activity of circulating antithrombin ( AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted similar to 20 angstrom, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.
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