Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates

Background: Understanding the networks of selective proteolysis that regula te complex biological systems requires an appreciation of the molecular mec hanisms used to maintain substrate specificity. Human plasmin, a serine pro tease that promotes the dissolution of blood clots and is essential in main taining normal hemostasis, is usually described as having broad substrate s pecificity. Recent evidence that plasmin also plays a key role in a variety of other important biological and pathological processes, however, has sug gested that this description might need to be re-evaluated. Results: We used substrate phage display to elucidate optimal subsite occup ancy for substrates of plasmin. We identified a peptide substrate that is c leaved 710,000-fold more efficiently by plasmin than a peptide containing t he activation sequence of plasminogen. Plasmin achieves this unexpected, la rge differential activity even though both target sequences possess an argi nine residue in the P1 position. We also demonstrate that proteolysis by pl asmin can be targeted to an engineered protein substrate and that introduct ion of substrate sequences identified by phage display into plasminogen inc reases plasmin-mediated cleavage of the mutant 2000-fold. Conclusions: The specificity of plasmin is more tightly controlled than pre viously recognized; interactions with substrates at all subsites between S4 and S2' contribute to catalysis. Furthermore, in contrast to most enzymes that exhibit positive selectivity for substrate, the evolution of substrate specificity by plasmin has apparently been dominated by a strong negative selection against development of autoactivation activity. This 'negative se lectivity' avoids short-circuiting regulation of the fibrinolytic system an d other important biological processes, and might be an important general m echanism for controlling protease cascades.