Conformational Activation of Zymogen-Like Thrombin Variants by Tight Binding Ligands

A series of well-established structural transitions lead to proteinase formation following zymogen cleavage in the family of serine proteinases homologous to chymotrypsin. These structural changes are triggered by the formation of a salt bridge following the generation of a new N-terminus that inserts in a sequence-specific way into a binding cleft. Based on the established dependence of proteinase maturation on the N-terminal sequence, we prepared a series of recombinant variants of human thrombin (IIa) in which the I¹⁶-V¹⁷-E¹⁸ N-terminal sequence in wild type IIa (IIa_WT) was replaced with I-G-E (IIa_IGE), T-A-T (IIa_TAT) or V-I-E (IIa_VIE). In comparison to IIa_WT, these variants exhibited greatly diminished ability to hydrolyze peptidyl substrates. Initial velocities measured with H-Dphenylalanyl- L-pipecolyl-L-arginine-p-nitroanilide (S2238) and L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroanilide (S2366) established a graded reduction in activity ranging from ~0.4% (IIa_IGE) to < 0.2% (IIa_VIE and IIa_TAT) that was also dependent on the concentration of the peptidyl substrate relative to Km. These findings are expected for variants that remain zymogen-like because of an impaired ability to drive proteinase formation. This interpretation was confirmed with studies using a fragment of staphylocoagulase (SC_1–325). Structural studies have established SC_1–325 to function as a conformational activator of the zymogen, prethrombin 2, that is accomplished by high affinity binding and the insertion of its N-terminus into the binding cleft. Saturating concentrations of SC_1–325 had a small inhibitory effect on IIa_WT but restored the rate of peptidyl substrate cleavage by IIa_TAT, IIa_VIE and IIa_IGE to levels observed with IIa_WT. While SC_1–325 could bind to prothrombin and prethrombin 2 with high affinity and yield catalytic activity, a deletion mutant of SC lacking 13 residues (SC_13–325) produced no detectable activity in the zymogen species. This result is consistent with the essential role played by the N-terminus of SC in conformational activation. SC_13–325 was found to bind to the series of IIa variants with inferred equilibrium dissociation constants of ~10⁻¹⁰M. Surprisingly, SC_13–325 restored peptidyl substrate cleavage by the zymogen-like variants of IIa to levels seen with IIa_WT. It follows that rescue of the zymogen-like variants of IIa does not require N-terminal insertion by SC. These inferences with peptidyl substrates were further pursued with binding studies examining the ability of N^α-dansyl-(p-guanidino)-L-phenylalanine-piperidide (I-2581) to bind to the active site. Fluorescence measurements indicated that I-2581 bound with high affinity to IIa_WT (Kd ~5 nM) but in an undetectably weak manner to IIa_IGE. However, saturating concentrations of SC_13–325 could completely restore high affinity binding of I-2581 to IIa_IGE. Thus, rescue of peptidyl substrate cleavage by SC_13–325 is matched by a restored ability of the zymogenlike variants to accommodate ligands at the active site. The findings raise the possibility that rescue of these variants by SC arises from ligand-dependent effects that stabilize proteinase structure and overcome the sub-optimal ability of I-G-E, T-A-T or V-I-E to insert properly into the binding cleft. This possibility was further investigated using the soluble extracellular domain of thrombomodulin (sTM) which represents one of the tightest binding natural ligands for IIa. As with SC_13–325, high concentrations of sTM were also found to enhance peptidyl substrate cleavage by IIa_IGE. Extent of rescue of IIa_IGE by sTM was also dependent on the substrate and the concentration of substrate relative to Km. In complex with sTM, IIa_IGE exhibited a partial restoration in protein C activation. While we have employed these N-terminal insertion variants as prototypic zymogen-like forms of IIa, rescue of these variants by strong ligands points to a generalized allosteric mechanism by which thrombin function may be regulated. Comparable or related phenomena may underlie the regulated action of thrombin on its range of biological substrates.