Tissue Factor Residues That Putatively Interact with Membrane Phospholipids

Ke Ke,James H Morrissey,Jian Yuan,Christopher B Doering

doi:10.1371/journal.pone.0088675

Abstract

Blood clotting is initiated by the two-subunit enzyme consisting of the plasma protease, factor VIIa (the catalytic subunit), bound to the integral membrane protein, tissue factor (the regulatory subunit). Molecular dynamics simulations have predicted that certain residues in the tissue factor ectodomain interact with phosphatidylserine headgroups to ensure optimal positioning of the tissue factor/factor VIIa complex relative to its membrane-bound protein substrates, factors IX and X. In this study, we individually mutated to alanine all the putative phosphatidylserine-interactive residues in the tissue factor ectodomain and measured their effects on tissue factor cofactor function (activation of factors IX and X by tissue factor/factor VIIa, and clotting of plasma). Some tissue factor mutants exhibited decreased activity in all three assays, with the most profound defects observed from mutations in or near the flexible loop from Lys159 to Gly164. The decreased activity of all of these tissue factor mutants could be partially or completely overcome by increasing the phosphatidylserine content of tissue factor-liposomes. Additionally, yeast surface display was used to screen a random library of tissue factor mutants for enhanced factor VIIa binding. Surprisingly, mutations at a single amino acid (Lys165) predominated, with the Lys165→Glu mutant exhibiting a 3-fold enhancement in factor VIIa binding affinity. Our studies reveal the functional contributions of residues in the C-terminal half of the tissue factor ectodomain that are implicated in interacting with phosphatidylserine headgroups to enhance tissue factor cofactor activity, possibly by allosterically modulating the conformation of the adjacent substrate-binding exosite region of tissue factor.

Highlights

Tissue factor (TF) is the cell-surface receptor for factor VIIa (FVIIa), with the resulting TF/FVIIa complex activating factors IX (FIX) and X (FX) by limited proteolysis [1]
Binding of FVIIa to membrane-anchored TF has multiple effects on enzyme activity: (a) TF allosterically activates FVIIa, independent of the membrane [2]; (b) TF is thought to contribute a substrate-binding site to help recognize FIX and FX [3,4,5,6]; (c) phosphatidylserine (PS) dramatically increases rates of FIX and FX activation by TF/FVIIa [7]; and (d) TF fixes FVIIa’s active site at a set distance above the membrane surface, which may be important for optimal engagement of the scissile bonds of FIX and FX [8,9,10,11]
This prompted us to hypothesize that PS-interacting residues in the TF ectodomain are in allosteric linkage with the nearby exosite region, possibly promoting a conformation better able to bind FIX and FX

Summary

Introduction

Tissue factor (TF) is the cell-surface receptor for factor VIIa (FVIIa), with the resulting TF/FVIIa complex activating factors IX (FIX) and X (FX) by limited proteolysis [1]. The TF residues predicted to interact with PS are located very close to the substrate-binding exosite region of this protein. This prompted us to hypothesize that PS-interacting residues in the TF ectodomain are in allosteric linkage with the nearby exosite region, possibly promoting a conformation better able to bind FIX and FX. We used mutagenesis to examine how TF residues predicted to interact with PS contribute to the proteolytic activity of the TF/FVIIa complex. We selected TF mutants with enhanced binding affinity to FVIIa. Our findings are consistent with the notion that conformational changes in TF, induced upon binding to both FVIIa and PS, may help regulate the catalytic activity of TF/FVIIa on membrane surfaces

Materials and Methods

Literature

Results and Discussion