Abstract
The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction approximately 2000-fold in the presence of phospholipid and Ca(2+). To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ(DeltaGD)) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing approximately 5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only approximately 2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional approximately 1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.
Highlights
Blood coagulation is a complex process requiring tight regulation through the use of feedback loops and the involvement of proteinase-specific inhibitors [1]. factor Xa (fXa),4 in complex with its cofactor Va, plays a crucial role in blood coagulation, being directly involved in the generation of the final proteinase thrombin from its zymogen prothrombin
We present here the structure of Z-dependent proteinase inhibitor (ZPI) in complex with Gla domainless-protein Z (PZ⌬GD), together with a model of the ternary complex with fXa that corresponds to the Michaelis complex
The P1 tyrosine results in a reaction that is several orders of magnitude slower than with P1 arginine, it ensures that reaction of ZPI with fXa only occurs at a significant rate, and as an inhibitor, when ZPI is membrane-associated through complexation with PZ
Summary
Blood coagulation is a complex process requiring tight regulation through the use of feedback loops and the involvement of proteinase-specific inhibitors [1]. fXa, in complex with its cofactor Va, plays a crucial role in blood coagulation, being directly involved in the generation of the final proteinase thrombin from its zymogen prothrombin. FXa, in complex with its cofactor Va, plays a crucial role in blood coagulation, being directly involved in the generation of the final proteinase thrombin from its zymogen prothrombin. To help prevent clot formation away from the site of injury, any fXa that dissociates from the membrane and diffuses away in the bloodstream must be efficiently inhibited This is accomplished primarily by the serpin antithrombin, which, when allosterically activated by being bound to heparan sulfate chains on the surface of endothelial cells, is a rapid inactivator of fXa, as well as of other circulating arginine-specific proteinases, such as thrombin and factor IXa [2]. The P1 tyrosine results in a reaction that is several orders of magnitude slower than with P1 arginine, it ensures that reaction of ZPI with fXa only occurs at a significant rate, and as an inhibitor, when ZPI is membrane-associated through complexation with PZ. This is another example of deliberate repression of a reaction unless compensating factors that ensure specificity (here for membrane-associated fXa) are present
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