Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa.
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.
Huang
Xin
X
Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Dementiev
Alexey
A
Olson
Steven T
ST
Gettins
Peter G W
PG
D001665:Binding Sites / Q000235:genetics; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001798:Blood Proteins; D010743:Phospholipids; C416370:SERPINA10 protein, human; D015843:Serpins; C026080:plasma protein Z; D018698:Glutamic Acid; D014443:Tyrosine; D015951:Factor Xa; D003545:Cysteine; D002118:Calcium