Abstract
The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.
Highlights
The proteolytically activated form of blood coagulation factor VII (FVII),2 factor VIIa (FVIIa), behaves zymogen-like with low enzymatic activity as opposed to other, constitutively active serine proteases of the coagulation cascade
FVIIaK161A, FVIIaD217A, and FVIIaD219A displayed a similar reduction in amidolytic activity whether bound to sTF or not
We have investigated the zymogenicity of FVIIa and factors that influence the insertion of the N terminus
Summary
The proteolytically activated form of blood coagulation factor VII (FVII), FVIIa, behaves zymogen-like with low enzymatic activity as opposed to other, constitutively active serine proteases of the coagulation cascade. We hypothesized that the effects of Ca2ϩ on FVIIa activity are through stabilization of the Ca2ϩ-binding loop, i.e. stabilization of the interaction(s) of Asp217/Asp219 with Lys161 in the N-terminal tail, and through alleviation of charge repulsion between the loop, primarily Asp212, and the nearby Glu296 The latter residue had been mutated in an earlier study [20] and the other four were replaced in the present study to obtain a complete set of variants to test the hypothesis. Our data suggest that Ca2ϩ binding to the 210 –220 loop in the protease domain of FVIIa promotes the intrinsic activity through stabilization of electrostatic interactions of Asp217 and Asp219 with Lys161 in the N-terminal tail and mitigation of the repulsion by Asp212 of Glu296 located close to loop 1 of the activation domain. Asp212 was identified as a new zymogenicity determinant, because the charge removal led to an increased intrinsic activity and degree of N terminus burial
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