Title : Basis for the specificity and activation of the serpin protein
Z-dependent proteinase inhibitor (
ZPI ) as an inhibitor of membrane-associated
factor Xa
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