Substitution of reactive centre loop residues from C1 esterase inhibitor increases the inhibitory specificity of alpha-1 antitrypsin for plasma kallikrein

C1 esterase inhibitor (C1INH) is a member of the serpin superfamily of proteins and controls plasma kallikrein (Pka). Purified C1INH concentrates are effective in controlling C1INH deficiency (hereditary angioedema, HAE). Because C1INH is a relatively slow inhibitor of Pka, we sought to develop a more effective inhibitor by exchanging reactive centre loop (RCL) residues in another serpin, alpha-1 antitrypsin (AAT) variant M358R, with the corresponding residues of C1INH. Novel, soluble, N-terminally hexahistidine-tagged variants were expressed in E. coli, purified by nickel chelate chromatography, and characterized kinetically. AAT/C1INH loop exchange mutants were designated by the RCL residues exchanged using the reactive centre P1-P1' convention. Maximal exchange mutant AC (10-4') inhibited Pka 78-fold and activated Factor XI (FXIa) 350-fold less rapidly than AAT M358R. Eleven additional variants were expressed, restoring AAT residues stepwise. The most selective variant was AC (10-3/4'), which restored AAT residues from P2-P3' compared to AC (10-4'), and inhibited Pka 1.9-fold more rapidly, and FXIa 1.6-fold less rapidly, for a gain in selectivity of 2.8-fold (p < 0.0001), without increasing the stoichiometry of inhibition (SI). The most active variant was AC (10-3), in which both the rate of Pka and FXIa inhibition were elevated relative to AAT M358R values, without SI elevation. Other variants exhibited slower reaction rates and/or elevated SI values. These results indicate that RCL exchanges can be productively employed to change serpin specificity and selectivity, but that the most effective exchanges may not be contiguous due to cooperativity between RCL residues.