Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold.

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule.

Why Ser and not Thr brokers catalysis in the trypsin fold.

Although Thr is equally represented as Ser in the human genome and as a nucleophile is as good as Ser, it is never found in the active site of the large family of trypsin-like proteases that utilize the Asp/His/Ser triad. The molecular basis of the preference of Ser over Thr in the trypsin fold was investigated with X-ray structures of the thrombin mutant S195T free and bound to an irreversible active site inhibitor. In the free form, the methyl group of T195 is oriented toward the incoming substrate in a conformation seemingly incompatible with productive binding.

Histone H4 promotes prothrombin autoactivation.

Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for proteolytic attack upon mutation of residues in the activation domain. Whether prothrombin autoactivation occurs in the wild-type under conditions relevant to physiology remains unknown. Here, we report that binding of histone H4 to prothrombin under physiological conditions generates thrombin by autoactivation.

Essential role of conformational selection in ligand binding.

Two competing and mutually exclusive mechanisms of ligand recognition - conformational selection and induced fit - have dominated our interpretation of ligand binding in biological macromolecules for almost six decades. Conformational selection posits the pre-existence of multiple conformations of the macromolecule from which the ligand selects the optimal one. Induced fit, on the other hand, postulates the existence of conformational rearrangements of the original conformation into an optimal one that are induced by binding of the ligand.

Conformational selection is a dominant mechanism of ligand binding.

Molecular recognition in biological macromolecules is achieved by binding interactions coupled to conformational transitions that precede or follow the binding step, two limiting mechanisms known as conformational selection and induced fit, respectively. Sorting out the contribution of these mechanisms to any binding interaction remains a challenging task of general interest in biochemistry. Here we show that conformational selection is associated with a vast repertoire of kinetic behaviors, can never be disproved a priori as a mechanism of ligand binding, and is sufficient to explain the relaxation kinetics documented experimentally for a large number of systems.

Conformational selection or induced fit? A critical appraisal of the kinetic mechanism.

For almost five decades, two competing mechanisms of ligand recognition, conformational selection and induced fit, have dominated our interpretation of ligand binding in biological macromolecules. When binding-dissociation events are fast compared to conformational transitions, the rate of approach to equilibrium, k(obs), becomes diagnostic of conformational selection or induced fit based on whether it decreases or increases, respectively, with the ligand concentration, [L]. However, this simple conclusion based on the rapid equilibrium approximation is not valid in general.

Conformational selection in trypsin-like proteases.

For over four decades, two competing mechanisms of ligand recognition--conformational selection and induced-fit--have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*).

Crystallographic and kinetic evidence of allostery in a trypsin-like protease.

Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E).

Evidence of the E*-E equilibrium from rapid kinetics of Na+ binding to activated protein C and factor Xa.

Na(+) binding to thrombin enhances the procoagulant and prothrombotic functions of the enzyme and obeys a mechanism that produces two kinetic phases: one fast (in the microsecond time scale) due to Na(+) binding to the low activity form E to produce the high activity form E:Na(+) and another considerably slower (in the millisecond time scale) that reflects a pre-equilibrium between E and the inactive form E*. In this study, we demonstrate that this mechanism also exists in other Na(+)-activated clotting proteases like factor Xa and activated protein C. These findings, along with recent structural data, suggest that the E*-E equilibrium is a general feature of the trypsin fold.