Crystallographic and Kinetic Evidence of Allostery in a Trypsin-like Protease.

The hallmark of allosteric proteins is that they exist in multiple conformations in equilibrium (1, 2). When alternative conformations differ in their functional properties, linkage is established between structure and biological activity and allostery becomes the basis of the effects observed experimentally. The theoretical underpinnings of allosteric transitions have been defined for systems working at equilibrium (1) or under transient kinetics (3). Yet structural validation of a pre-existing equilibrium between alternative conformations remains a challenge even for textbook examples of allosteric proteins (2, 4). Allostery is not an exclusive property of multimeric proteins. Indeed, the ability of monomeric enzymes to express complex behavior consistent with allosteric transitions has long been recognized (3, 5). Trypsin-like proteases are monomeric enzymes which constitute the largest and best studied group of homologous proteases in the human genome (6). They are phylogenetically grouped into six functional categories: digestion, coagulation and immunity, tryptase, matriptase, kallikrein and granzymes. Trypsin-like proteases share a common mechanism of catalysis that relies upon the coordinate action of three catalytic residues: H57, D102 and S195 (chymotrypsinogen numbering). In addition, they share a common mechanism of activation: an inactive zymogen precursor is proteolytically cut between residues 15 and 16 to generate a new N-terminus that ion-pairs with the highly conserved D194 next to the catalytic S195 and organizes both the oxyanion hole and primary specificity pocket (6–8). The irreversible zymogen→protease conversion affords a useful paradigm to explain the onset of catalytic activity as seen in the digestive system, blood coagulation or the complement system and is particularly useful to understand the initiation, progression and amplification of enzyme cascades, where each component acts as a substrate in the inactive zymogen form in one step and as an active enzyme in the subsequent step (9). However, considerable variation in catalytic activity is observed among members of the trypsin family following conversion from the inactive zymogen form. Digestive enzymes like trypsin, chymotrypsin and elastase, complement factors C1r and C1s, and coagulation factors like thrombin are highly active after the zymogen→protease conversion has taken place. On the other hand, complement factors B and C2 are mostly inactive until binding of complement factors C3b and C4b enable catalytic activity at the site where amplification of C3 activation leads to formation of the membrane attack complex (10–12). Coagulation factor VIIa circulates in the blood as a poorly active protease that acquires full catalytic competence only upon interaction with tissue factor that becomes exposed to the blood stream upon vascular injury (13, 14). Complement factor D assumes an inactive conformation with a distorted catalytic triad (15, 16) until binding to C3b and factor B promote substrate binding and catalytic activity (17, 18). The high catalytic activity of trypsin, C1r and thrombin and the ability of complement factor D or coagulation factor VIIa to remain in a zymogen-like form suggests that the trypsin fold may assume active and inactive conformations even after the zymogen→protease conversion has taken place. Existence of an allosteric equilibrium between active and inactive forms has been proposed for coagulation factor VIIa (14, 19). Rapid kinetics support a pre-existing equilibrium between active (E) and inactive (E*) forms for thrombin (20, 21), meizothrombin desF1 (22), factor Xa and activated protein C (23). Structures of thrombin in the free form reveal a conformation, E, with the active site open (24–26) and an alternative conformation, E*, with the active site blocked by repositioning of the 215–217 segment (27–30). However, no evidence currently exists that the same protease construct may assume alternative conformations that can be trapped by crystallographic analysis. This evidence is reported here for the first time.