Interplay between conformational selection and zymogen activation

Abstract

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.

19F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2

“…The mechanism of recognition for small tripeptides is consistent with a pre-existing E*-E equilibrium of conformational selection rather than induced fit (10,14). The E* form prevails in the zymogen and gradually shifts to the E form during transition to the mature protease (15). In fact, the E*-E equilibrium complements the Huber-Bode mechanism and contributes to organization of the active site for efficient binding and catalysis (16).…”

“…The Huber-Bode mechanism also appears not to be sufficient for protease function. A pre-existing equilibrium between closed (E*) and open (E) conformations of the active site controls the onset of substrate binding and catalysis (15). In the E* form, substrate cannot bind to the active site, and catalysis is impeded.…”

“…Although evidence of the E*-E equilibrium from the current structural database (9,17,18) and rapid kinetics (10,14,15,17) is solid, direct proof that such conformational plasticity exists in solution remains elusive. Structural features revealed by X-ray may be biased by crystal packing or by the extreme solution conditions often necessary to achieve crystallization.…”

“…From these studies, Huntington (21) and Krishnaswamy and co-workers (22) have concluded that thrombin is inherently plastic and shuttles within an ensemble of conformations that are disordered and zymogen-like when free but rigid and protease-like when bound (21,22). However, this ensemble view of thrombin (20 -22) remains a speculation largely inconsistent with existing X-ray (9,17,18) and rapid kinetics (10,14,15,17) data and lacks validation from NMR studies of free thrombin or of the zymogen precursor prethrombin-2. Whether free thrombin is zymogen-like and switches to the mature conformation by induced fit or pre-exists in alternative conformations from which the ligand selects the optimal fit can only be established by studies of the free form.…”

“…We labeled all nine Trp residues in thrombin and prethrombin-2 to interrogate the conformational properties of the protein in the absence of any added ligand and Na ϩ . 19 F NMR has been used for a number of biologically important systems (25)(26)(27)(28)(29) and is ideally suited for studying the Trp residues of thrombin that are known to report on changes linked to the E*-E equilibrium, the zymogen-toprotease conversion, and ligand binding (14,15,30).…”