Driving Thrombin From a Protease-Like State to a Zymogen-Like State and Back.
Authors: Kamath Parvathi , Krishnaswamy Sriram 
Published: October 2019

Abstract 852

Thrombin (IIa) possesses two anion binding exosites, ABEI and ABEII that play key roles in binding ligands and regulating protease function. Thrombomodulin and hirugen (Hir) are among the established ligands for ABEI. ABEII binds to fragment 1.2 (F12), the activation peptide produced upon conversion of prothrombin to IIa. ABEI ligands bind weakly to the zymogen in comparison to the protease. In contrast, recent findings suggest that F12, the authentic ABEII ligand, binds the zymogen with higher affinity than it does the protease. This reciprocal relationship in ligand binding upon conversion of the zymogen to protease was investigated using a series of reference states expected to be positioned differentially along the pathway for conversion of zymogen to protease. IIaS195A, with S195 mutated to A to render it catalytically inactive, was used to represent the protease with an unligated active site. Its uncleaved precursor, prethrombin 2 (P2S195A), was the zymogen. The variant IIaTAT, with residues I16V17E18 mutated to T16A17T18 is known to be a zymogen-like form of IIa. Covalent inhibition of IIa with Phe-Pro-Arg-chloromethyl ketone to yield IIai, locked it in an ultimate protease state. The binding of F12 to these species was studied at physiologic pH and ionic strength by isothermal titration calorimetry, to provide unambiguous and direct assessment of the thermodynamic parameters associated with the ligation of ABEII. The interaction of F12 with P2S195A was most thermodynamically favorable. There was a systematic change in thermodynamic parameters as the protease-like nature of the IIa variant increased. While changes in ΔG were modest, changes in ΔH and ΔS were dramatic and compensatory (ΔΔH= +15.6 kcal.mol-1 and ΔTΔS= +13.56 kcal.mol-1). These large compensating changes yielded a linear relationship between ΔH and ΔS, expected for different states in reversible equilibrium with each other. The reference species define a continuum of states, poised between zymogen and protease, which can be distinguished on the basis of thermodynamic constants for the ligation of ABEII by F12. Binding of F12 to the zymogen-like IIaTAT in the presence of increasing fixed concentrations of a tight binding active site ligand produced a shift in thermodynamic constants to those observed with IIai. This could be analyzed by a system of equilibria indicating that the active site ligand could drive IIaTAT to the fully stabilized protease-like state with inferred Kd= 18.3 ± 2.8 μM in agreement with the independently measured value of 31.8 ± 2.2 μM. Na+ binding is also established to modulate IIa function. Binding of F12 to IIaS195A, at constant ionic strength but with decreasing concentrations of Na+, systematically shifted the thermodynamic constants to those observed with the zymogen-like IIaTAT. Thus, reduced saturation with Na+ transitions IIaS195A towards a zymogen-like state. Analysis of this family of titration curves yielded Kd=8.3 ± 0.4 mM for Na+ binding to IIaS195A in agreement with Kd=4.4 ± 0.34 mM for its binding to IIa inferred from kinetic studies. In opposition, Hir, the ABEI ligand, bound the protease-like reference species more favorably than the zymogen-like forms. Decreasing concentrations of Na+ systematically weakened Hir binding to IIaS195A to approach that seen with IIaTAT. Analysis of these titration curves were consistent with a system of equilibria in which the zymogen-like species that predominates in the absence of Na+ can be forced into a protease-like state by saturating ABEI. These findings illustrate the opposing effects that the conversion of zymogen to protease has on ABEI and ABEII function. They provide independent lines of support for a network of thermodynamic linkage, not always evident from measurement of affinity (or ΔG), between ABEI, ABEII, the active site as well as the Na+ site. The basis for this linkage, only revealed by direct thermodynamic measurement, lies in the ability of IIa to interconvert, in a ligand-dependent manner, between zymogen-like and protease-like states. Such conformational plasticity, driven by ligand binding, likely plays a major role in the regulation of the diverse functions of IIa.

Disclosures:

No relevant conflicts of interest to declare.