Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry

TY - JOUR

T1 - Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry

AU - Song, Hongjian

AU - Olsen, Ole H

AU - Persson, Egon

AU - Rand, Kasper Dyrberg

N1 - Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

PY - 2014/12/19

Y1 - 2014/12/19

N2 - Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease.

AB - Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease.

U2 - 10.1074/jbc.M114.614297

DO - 10.1074/jbc.M114.614297

M3 - Journal article

C2 - 25344622

SN - 0021-9258

VL - 289

SP - 35388

EP - 35396

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

ER -