Long-Range Allosteric Linkage Between Exosites Reciprocally Regulates the Zymogenicity of Prothrombin Derivatives

The binding of ligands to anion binding exosite I (ABEI) and exosite II (ABEII) on prothrombin (II) derivatives plays an integral role in regulating their function. These exosites are located on opposite faces of the proteinase domain and exhibit unique binding specificities. Fragment 1.2 (F12) binds to ABEII and acts as a zymogen-promoting allosteric ligand. Conversely, Na + and active site ligands stabilize the proteinase state. Here, we investigated the allosteric linkage between ABEI and the Na + binding site, active site or ABEII using multiple ABEI ligands and prothrombin derivatives differentially poised along the zymogen to proteinase continuum. Prethrombin 2 (P2) represents the most zymogen-like state that differs from thrombin (IIa) because it is not cleaved at the R 320 site. To mimic the zymogen-like character of P2 in a cleaved IIa molecule, residues responsible for N-terminal insertion and proteinase formation (IVE) were swapped with TAT to produce IIa TAT , a mutant with vastly diminished proteolytic activity. Alanine was substituted with the catalytic serine residue in IIa (IIa S195A ) to represent the proteinase without a ligand at its active site. The thermodynamics of interactions between the thrombin inhibitor and ABEI ligand hirugen (Hir) and the various reference states was assessed using isothermal titration calorimetry (ITC). Titration of Hir into P2, IIa TAT or IIa S195A revealed thermodynamically more favorable binding to proteinase-like IIa S195A in comparison to zymogen-like P2 or IIa TAT . Binding of Hir to IIa S195A was affected by the concentration of Na + at constant ionic strength. Global analysis done in the presence of increasing concentrations of Na + revealed a 5-fold increase in Hir binding affinity when IIa S195A is ligated with Na + ; demonstrating positive allosteric linkage between ABEI and Na + binding. Using a truncated Staphylocoagulase variant that binds ABEI without N-terminus insertion (SC 13-325 ), we found that SC 13-325 binding alone promoted active site opening and fluorescent inhibitor incorporation in zymogen-like P2. These data reinforce the observation that ABEI ligands promote a proteinase-like state in prothrombin derivatives through positive allosteric linkage with the Na + binding and active sites. Interestingly, inhibitor incorporation and ITC studies both showed that SC 13-325 interacts poorly with II, but strongly with P2 despite both species being zymogens. These findings imply that the ABEII ligand F12, which is produced upon cleavage of II at R 271 to form P2, may display negative allosteric linkage with ABEI. Titration of SC 13-325 into pre-formed complexes of P2/F12 revealed a drastic reduction in affinity of SC 13-325 for P2 when F12 is bound to ABEII. Thus, F12 binding at ABEII negatively affects ABEI binding. Further studies used soluble thrombomodulin (sTM) as the most physiologically pertinent ligand for ABEI. ITC and global analysis of the binding of F12 to P2 in the presence of different concentrations of sTM revealed a ~1200-fold decrease in binding affinity and enthalpy for either the binding of F12 to P2 bound to sTM or the binding of sTM to P2 bound to F12. These data illustrate the strong negative linkage associated with the binding of protein ligands to ABEI and ABEII which yields the appearance of competitive and mutually exclusive binding at the two sites despite the fact that they are on opposite sides of the proteinase domain. Allosteric linkage between ligand binding at the two exosites is centered on the ability of F12 binding to ABEII and favor zymogen-like forms and ligand binding to ABEI to favor proteinase-like forms. Thus, allosteric linkage among exosites is vital to the interconversion of prothrombin species along the zymogen to proteinase spectrum. These ligand-dependent conformational shifts and associated changes in function are likely to greatly contribute to the dynamic roles that IIa plays during coagulation. Disclosures No relevant conflicts of interest to declare.