Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the K of 1.0 nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions-the deprotonation of the sulfonamide amino group, the protonation of CA-Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design.

Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E*) to binding. Prothrombin exists mainly in the E* conformational ensemble and conversion to thrombin produces two dominant changes: a progressive shift toward the E conformational ensemble triggered by removal of the auxiliary domains upon cleavage at R271 and a drastic drop of the rate of ligand dissociation from the active site triggered by cleavage at R320. Together, these effects produce a significant (700-fold) increase in binding affinity. Limited proteolysis reveals how the E*-E equilibrium shifts during prothrombin activation and influences exposure of the sites of cleavage at R271 and R320. These new findings on the molecular underpinnings of prothrombin activation are relevant to other zymogens with modular assembly involved in blood coagulation, complement and fibrinolysis.

The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention.

Conformational selection (CS) and induced fit (IF) are two widely used interpretations of binding of a ligand to biological macromolecules. Both mechanisms envision a two-step reaction in which a conformational transition either precedes (CS) or follows (IF) the binding step. Under pseudo-first-order conditions where the ligand is in excess compared to the macromolecule, both mechanisms produce two relaxations. A fast one eventually increases linearly with ligand concentration and reflects the binding interaction. A slow one saturates to a constant value after decreasing or increasing hyperbolically with ligand concentration. This relaxation is the one most often accessible to experimental measurements and is potentially diagnostic of the mechanism involved. A relaxation that decreases unequivocally identifies CS, but a hyperbolic increase is compatible with both CS and IF. The potential ambiguity between the two mechanisms is more than qualitative. Here we show that the entire kinetic repertoire of IF is nothing but a mathematical special case of CS as revealed by a simple transformation of the rate constants, which emphasizes the need for independent support of either mechanism from additional experimental evidence. We discuss a simple strategy for distinguishing between IF and CS under the most common conditions encountered in practice, i.e., when the ligand is in excess compared to the macromolecule and a single relaxation is accessible to experimental measurements.

In recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.