Abstract
BackgroundAlpha-1 proteinase inhibitor (API) is a plasma serpin superfamily member that inhibits neutrophil elastase; variant API M358R inhibits thrombin and activated protein C (APC). Fusing residues 1-75 of another serpin, heparin cofactor II (HCII), to API M358R (in HAPI M358R) was previously shown to accelerate thrombin inhibition over API M358R by conferring thrombin exosite 1 binding properties. We hypothesized that replacing HCII 1-75 region with the 13 C-terminal residues (triskaidecapeptide) of hirudin variant 3 (HV354-66) would further enhance the inhibitory potency of API M358R fusion proteins. We therefore expressed HV3API M358R (HV354-66 fused to API M358R) and HV3API RCL5 (HV354-66 fused to API F352A/L353V/E354V/A355I/I356A/I460L/M358R) API M358R) as N-terminally hexahistidine-tagged polypeptides in E. coli.ResultsHV3API M358R inhibited thrombin 3.3-fold more rapidly than API M358R; for HV3API RCL5 the rate enhancement was 1.9-fold versus API RCL5; neither protein inhibited thrombin as rapidly as HAPI M358R. While the thrombin/Activated Protein C rate constant ratio was 77-fold higher for HV3API RCL5 than for HV3API M358R, most of the increased specificity derived from the API F352A/L353V/E354V/A355I/I356A/I460L API RCL 5 mutations, since API RCL5 remained 3-fold more specific than HV3API RCL5. An HV3 54-66 peptide doubled the Thrombin Clotting Time (TCT) and halved the binding of thrombin to immobilized HCII 1-75 at lower concentrations than free HCII 1-75. HV3API RCL5 bound active site-inhibited FPR-chloromethyl ketone-thrombin more effectively than HAPI RCL5. Transferring the position of the fused HV3 triskaidecapeptide to the C-terminus of API M358R decreased the rate of thrombin inhibition relative to that mediated by HV3API M358R by 11-to 14-fold.ConclusionsFusing the C-terminal triskaidecapeptide of HV3 to API M358R-containing serpins significantly increased their effectiveness as thrombin inhibitors, but the enhancement was less than that seen in HCII 1-75–API M358R fusion proteins. HCII 1-75 was a superior fusion partner, in spite of the greater affinity of the HV3 triskaidecapeptide, manifested both in isolated and API-fused form, for thrombin exosite 1. Our results suggest that HCII 1-75 binds thrombin exosite 1 and orients the attached serpin scaffold for more efficient interaction with the active site of thrombin than the HV3 triskaidecapeptide.
Highlights
Alpha-1 proteinase inhibitor (API) is a plasma serpin superfamily member that inhibits neutrophil elastase; variant Alpha-1-proteinase inhibitor (API) in N-to Cterminal order (M358R) inhibits thrombin and activated protein C (APC)
Induction of bacterial cultures harbouring expression plasmids specifying HV3API M358R with arabinose led to the appearance of a novel 47 kDa protein in total soluble lysate preparations, that was enriched by nickel-chelate chromatography and purified following an additional ion exchange step, like API M358R and HAPI M358R
This approach increased the rate of inhibition of αthrombin inhibition both for HV3API M358R, and for HV3API HAPI M358R with additional F352A/L353V/E354V/A355I/I356A/I460L substitutions (RCL5), in which six reactive centre loop (RCL) residues in addition to M358R were mutated to the corresponding residues in antithrombin
Summary
Alpha-1 proteinase inhibitor (API) is a plasma serpin superfamily member that inhibits neutrophil elastase; variant API M358R inhibits thrombin and activated protein C (APC). API inhibits other serine proteinases such as thrombin [7], coagulation factor XIa [8], and activated protein C (APC) [9], but at rates at least six orders of magnitude less rapid than its inhibition of elastase; it efficiently inhibits trypsin, but would not be expected to come in contact with this digestive system enzyme under physiological conditions Serpins inhibit their cognate proteinases via a complex mechanism initiated when the reactive centre loop (RCL) surface structure forms an encounter complex with the active site of a protease [2]. Crystal structures of API in intact form [16], in RCLcleaved form [17], in encounter complexes with trypsin [18], and in covalent serpin-enzyme complexes with trypsin [15] and elastase [14] have contributed to the elucidation of this mechanism
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