Allostery in trypsin-like proteases suggests new therapeutic strategies

Abstract

Trypsin-like proteases are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. A current paradigm posits that the irreversible transition from an inactive zymogen to the active protease form enables productive interaction with substrate and catalysis. Analysis of the entire structural database reveals two distinct conformations of the active site: one fully accessible to substrate (E) and the other occluded by the collapse of a specific segment (E*). The allosteric E*-E equilibrium provides a reversible mechanism for activity and regulation besides the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. Here, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.

Crystal Structure of Prothrombin Reveals Conformational Flexibility and Mechanism of Activation

“…5). In addition, access to the primary specificity pocket is hindered by collapse of the 547-549 (215-217) segment, a signature of the inactive E* form of the protease and zymogen (43,44) that brings Trp-547 (Trp-215) in hydrophobic interaction with Trp-370 (Trp-60d) in the 60-loop and Trp-468 (Trp-148) in the autolysis loop (Fig. 5).…”

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.…”

Autoactivation of Thrombin Precursors

“…In the case of chymotrypsinogen (18) and prethrombin-2 (23), the immediate zymogen precursor of the clotting protease thrombin, alternative conformations of the 215-217 segment consistent with a pre-existing E*-E equilibrium in solution have been trapped in the same crystal structure or different crystals harvested from the same crystallization well. The equilibrium between active (E) and inactive (E*) forms is a basic property of the trypsin fold for both the protease and zymogen (21,22) and provides a relevant example of conformational selection as the basis of functional and structural plasticity in proteins (24,25). Conformational selection in terms of the E*-E equilibrium is the only ligand binding mechanism that can be assigned unambiguously from rapid kinetics data (26) and was originally reported for chymotrypsin Ͼ40 years ago (27).…”

“…A number of zymogens crystallize in a conformation (E* form) with the active site occluded by collapse of the 215-217 segment, the oxyanion hole incorrectly formed, and residues of the catalytic triad not optimally aligned for H-bonding interaction. Other zymogens such as trypsinogen (3,16), the zymogen of MASP-2 (17), chymotrypsinogen (18), coagulation factor XI (19), and complement profactor B (20) crystallize in an alternative conformation (E form) where the active site is fully accessible to substrate and organized as in the mature protease (21,22). In the case of chymotrypsinogen (18) and prethrombin-2 (23), the immediate zymogen precursor of the clotting protease thrombin, alternative conformations of the 215-217 segment consistent with a pre-existing E*-E equilibrium in solution have been trapped in the same crystal structure or different crystals harvested from the same crystallization well.…”