Exosites, ligands and complex formation: novel determinants of protease specificity

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Proteins are one of the three building blocks of life and there are at least 100,000 different of them in our body. During their life-time, all of these proteins will encounter protein-degrading enzymes, called proteases, which will either lead to their activation, inactivation, modification or complete removal. Proteolysis, the degradation of proteins, is thus a fundamental process and must be tightly regulated. A random degradation of proteins would lead to severe cell and tissue damage. The selective protein degradation is accomplished by an army of about 700 different proteases. How do all these proteases recognize their protein substrates? Our research proposal aims to answer this question. As proteins can be very large, proteases do likely recognize certain surface features on their target substrates which can be remote from the actual cleavage site. We have identified so-called exosites on elastin-degrading proteases, which specifically interact with this protein. Without the binding of elastin to the exosites, no degradation occurs. We also hypothesize that these exosite protein interactions situate the substrate into the right orientation to allow cleavage or even modulate the structure of the substrate to make cleavage easier. Elastin is one of the most abundant but also most protease-resistant proteins in our bodies and is responsible for the integrity and elasticity of our organs including arteries, lung and skin. Interestingly, the same exosites are also used by another protease to form a protease complex which specifically degrades collagen, another major structural protein. This tells us that the same or similar exosites can be exploited for the degradation of different proteins using different mechanisms. Using biochemical, structural, and cellular approaches, we will characterize the nature and architectures of those exosites and explore novel mechanisms of selective protein degradation. We will also characterize certain ligands which interact with exosites and regulate the substrate specificity of these proteases.