Allostery in trypsin-like proteases suggests new therapeutic strategies

Abstract

Trypsin-like proteases (TLPs) 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 in addition to the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. In this review, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.

Figure 1

Structure of the active site of a representative trypsin-like protease. Shown are the catalytic residues (H57, D102, S195), the primary specificity site (D189), the oxyanion hole formed by the backbone N atoms of S195 and G193 (red arrow), residues of the 215-217 segment and the H-bond between the N-terminus of the catalytic chain (I16) and the side chain of D194. Catalysis requires correct folding of the active site promoted by activation of the zymogen and formation of the I16-D194 H-bond, correct positioning of the catalytic residues for H transfer, correct architecture of the oxyanion hole. Binding of substrate is optimized by interaction with the primary specificity pocket (residue 189) and docking against the 215-217 segment.

Figure 2

Active site accessibility in the protease and zymogen. Accessibility of the active site for all proteases in the set (Box 1) was calculated in terms of the overlap with PPACK bound to the active site in the structure 1SHH of thrombin and expressed as percentage of the total volume of inhibitor. The structures in the set are all free of ligands bound to the active site or at sites known to affect activity or stability and therefore provide relevant sampling of the conformations accessible to the fold in the free form. The analysis identifies two groups in each set: one with considerable blockage of the active site where overlap with the volume to be occupied by PPACK is on the average 119 ų (36% of total, red bars), and the other with negligible or no overlap averaging 1.0 ų (0.3% of total, green bars). The dichotomous distribution supports existence of an equilibrium between mutually exclusive, collapsed and open forms in both the zymogen and protease.

Figure 3

Zymogen to protease conversion scheme. The classical zymogen to protease conversion scheme is extended to account for allostery in the zymogen and protease. Two forms of the zymogen, one with the active site open (Z) and the other with the active site collapsed (Z*) are in equilibrium and each converts irreversibly to the E or E* form of the protease. Both Z and Z* are inactive. Activity of the protease depends on the distribution between the inactive E* and active E form. The Z*-Z equilibrium in the zymogen controls the rate of conversion to the protease. Natural cofactors and synthetic molecules affect activity of the protease or the zymogen to protease conversion by altering the distribution of the various species in the scheme, as discussed in the text.

Figure 4

Crystal structures of the anticoagulant thrombin mutants WE [(a) 1TQ0] and Δ146-149e [(b) 3GIC]. The structures are overlayed with the structure of thrombin bound to PPACK (green, 1SHH) where only PPACK is shown for clarity. The 215-217 segment carrying the W215A/E217A mutation collapses into the active site and clashes with the side chain of Arg at the P1 position of PPACK occluding 29% of the total volume of the inhibitor (311 ų). In the case of Δ146-149e, the entire 215-217 moves into the active site and occludes 48% of the volume of PPACK. The structures of WE and Δ146-149e offer substantial insight into the mechanism of action of these mutants and show how thrombin can be turned into an anticoagulant with site-directed mutations that stabilize the E* form.

Figure I

The E and E* forms. Overlay of the structures of thrombin in the E (1SGI, green) (35) and E* (3BEI, magenta) (38) form on the structure of thrombin bound to the active site inhibitor PPACK (1SHH, white) (35). PPACK (yellow) and residues H57, S195 and W215 from the protein are rendered as sticks. In the E form there is no overlap of residues 215-217 with the space occupied by PPACK in the active site. In the E* form, collapse of W215 and the 215-217 segment produces 159 ų occlusion of the space to be occupied by PPACK, which represents 48% of the total volume of the inhibitor (331 ų).