Serine Protease Inhibitors - Conformational Modulation of Serpin Activity

Conformational Modulation of Serpin Activity

The conformational mobility of serpins provides a key advantage over static lock-and-key protease inhibitors. In particular, the function of inhibitory serpins can be readily controlled by specific cofactors. The X-ray crystal structures of antithrombin, heparin cofactor II, MENT and murine antichymotrypsin reveal that these serpins adopt a conformation wherein the first two amino acids of the RCL are inserted into the top of the A β-sheet (see figures 4 and 7). The partially inserted conformation is important because co-factors are able to conformationally switch certain partially inserted serpins into a fully expelled form. This conformational rearrangement makes the serpin a more effective inhibitor.

The archetypal example of this situation is antithrombin, which circulates in plasma in a partially inserted relatively inactive state. The primary specificity determining residue (the P1 Arginine) points toward the body of the serpin and is unavailable to the protease (Figure 7). Upon binding a high-affinity heparin pentasaccharide sequence within long-chain heparin, antithrombin undergoes a conformational change, RCL expulsion, and exposure of the P1 Arginine. The heparin pentasaccharide-bound form of antithrombin is, thus, a more effective inhibitor of thrombin and factor Xa (figure 7). Furthermore, both of these coagulation proteases contain binding sites (called exosites) for heparin. Heparin, therefore, also acts as a template for binding of both protease and serpin, further dramatically accelerating the interaction between the two parties (Figure 7). After the initial interaction, the final serpin complex is formed and the heparin moiety is released. This interaction is physiologically important. For example, after injury to the blood vessel wall, heparin is exposed, and antithrombin is activated to control the clotting response. The understanding of the molecular basis of this interaction formed the basis of the development of Fondaparinux, a synthetic form of Heparin pentasaccharide used as an anti-clotting drug.

Certain serpins spontaneously undergo the S-to-R transition as part of their function, to form a conformation termed the latent state (Figure 8). In latent serpins, the first strand of the C-sheet has to peel off to allow full RCL insertion. Latent serpins are unable to interact with proteases and are not protease inhibitors. The transition to latency represents a control mechanism for the serpin PAI-1. PAI-1 is released in the inhibitory conformation, however, undergoes conformational change to the latent state unless it is bound to the cofactor vitronectin. Thus PAI-1 contains an "auto-inactivation" mechanism. Similarly, antithrombin can also spontaneously convert to the latent state as part of its normal function. Finally, the N-terminus of tengpin (see pdbs 2PEE and 2PEF), a serpin from Thermoanaerobacter tengcongensis, is required to lock the molecule in the native inhibitory state. Disruption of interactions made by the N-terminal region results in spontaneous conformational change of this serpin to the latent conformation.