Conformational selection in trypsin-like proteases

Abstract

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*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics.

Histone H4 Promotes Prothrombin Autoactivation

“…Autoactivation is found in other zymogens such as proprotein convertases furin and kexin type 9 (31-33), plasma hyaluronan-binding protein (26), recombinant factor VII (34), and the membrane-bound matriptases (35,36). The pre-existing equilibrium of the trypsin fold between the E* (inactive) and E (active) forms (7,37) explains autoactivation in terms of the small intrinsic activity of the zymogen (6). However, in the case of prothrombin, the question remains as to the physiological relevance of autoactivation given that Arg-320 (Arg-15) is not accessible to proteolytic attack in the wild-type (5).…”

“…Whether a linkage exists in prothrombin between the E*-E equilibrium affecting the active site region (7,37,42) and the equilibrium between the collapsed and fully extended conformations made possible by the flexibility of the linker between kringle-1 and kringle-2 (5) remains to be established. The possibility is intriguing as it suggests a long range communication between the catalytic domain of the zymogen and its auxiliary domains, thereby offering a much needed structural perspective on the mechanism of prothrombin activation.…”

“…The conversion typically unfolds over several hours, but in some cases, it is extremely rapid and completes during purification (6). The conversion is initiated by the zymogen itself by virtue of the pre-existing equilibrium between inactive (E*) and active (E) forms (7) and is then propagated and amplified by the mature enzyme. Whether autoactivation takes place in the wild-type protein under the influence of specific cofactors, as observed in other zymogens (8,9), remains an unresolved issue of potential physiological significance because it would afford a mechanism of thrombin generation that bypasses activation of the coagulation cascade.…”

A Monoclonal Antibody (MCPR3-7) Interfering with the Activity of Proteinase 3 by an Allosteric Mechanism

“…The activation domain of mature PR3 can adopt a zymogen-like conformation (E*) even after propeptide removal although with low probability (24). Binding of an antibody could forcefully induce this zymogenlike, catalytically inactive state even in mature, amino-terminally processed PR3, resulting in a loss of the S1 binding site and oxyanion hole.…”

“…Allosteric regulation of the two segments around residues 190 and 220 essentially switches the molecule from a functionally incompetent zymogen into a catalytically competent state and creates the S1 binding site and oxyanion hole (22,23). This conformational switch between the proenzyme state and the catalytically active state of the mature enzyme is the well established structural basis for allosteric regulation of trypsin-/chymotrypsin-like enzyme activity (24).…”