Abstract
We have previously shown that residues Tyr-253 and Glu-255 in the serpin antithrombin function as exosites to promote the inhibition of factor Xa and factor IXa when the serpin is conformationally activated by heparin. Here we show that functional exosites can be engineered at homologous positions in a P1 Arg variant of the serpin alpha1-proteinase inhibitor (alpha1PI) that does not require heparin for activation. The combined effect of the two exosites increased the association rate constant for the reactions of alpha1PI with factors Xa and IXa 11-14-fold, comparable with their rate-enhancing effects on the reactions of heparin-activated antithrombin with these proteases. The effects of the engineered exosites were specific, alpha1PI inhibitor reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in factor Xa, which interacts with the exosite residues in heparin-activated antithrombin, abrogated the ability of the engineered exosites in alpha1PI to promote factor Xa inhibition. Binding studies showed that the exosites enhance the Michaelis complex interaction of alpha1PI with S195A factor Xa as they do with the heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP reactive loop residues in the alpha1PI exosite variant with a preferred IEG substrate sequence for factor Xa modestly enhanced the reactivity of the exosite mutant inhibitor with factor Xa by approximately 2-fold but greatly increased the selectivity of alpha1PI for inhibiting factor Xa over thrombin by approximately 1000-fold. Together, these results show that a specific and selective inhibitor of factor Xa can be engineered by incorporating factor Xa exosite and reactive site recognition determinants in a serpin.
Highlights
The ubiquitous proteins of the serpin superfamily share a common structure and mostly function as inhibitors of intracellular and extracellular serine and cysteine-type proteases in a vast array of physiologic processes [1, 2]
Engineering Antithrombin Exosites in ␣1PI—Two residues in antithrombin, Tyr-253 and Glu-255 in strand 3 of -sheet C, are principal determinants of an exosite that promotes the binding of the target proteases, factor Xa and factor IXa, when antithrombin is conformationally activated by heparin [9]
Assuming a stoichiometry of 1 and a maximum fluorescence change equal to that obtained with the titration with the double mutant, a KD of ϳ70 M was estimated for the interaction of S195A factor Xa with the wild-type ␣1PI. To confirm these differences in binding affinity, we examined the ability of S195A factor Xa to competitively inhibit the reaction of active factor Xa with wild-type and double exosite mutant ␣1PIs
Summary
Proteins—Recombinant ␣1PI exosite mutants were produced from an ␣1PI background containing two mutations, a P1 Met358 change to Arg and a Cys-232 change to Ser, as in past studies [17]. ␣1PI variants were produced in Escherichia coli BL21 cells using a T7 expression system from Invitrogen and refolded from inclusion bodies as described [17, 18]. Reactions of factor Xa and trypsin with wild-type and variant ␣1PIs were monitored continuously in the presence of a reporter chromogenic substrate by following the time-dependent decrease in rate of substrate hydrolysis at 405 nm as in previous studies. In the case of reactions of thrombin and factor IXa with wild-type and variant ␣1PIs, kobs values were obtained from exponential fits of the decrease of active protease with time measured by discontinuous assays of residual protease activity. After incubating for 1 min (␣1PI double exosite mutant) or for 10 min (␣1PI wild type), a time yielding 90% factor Xa inhibition in the absence of S195A protease, the remaining proteolytic activity was determined by adding factor Xa substrate solution and measuring the initial rate of substrate hydrolysis. Where kobs,o and kobs represent kobs values measured in the absence and presence of S195A factor Xa, respectively, and FXa represents factor Xa
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