The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition
Journal of Biological Chemistry
The Kunitz-STI family is a paradigm of protease-inhibitor interaction in particular and protein-protein recognition in general. Results: PPI is a versatile protease inhibitor that targets several subfamilies of serine proteases. Conclusion: The ␤-trefoil fold constitutes an evolutionary platform for protease inhibition and molecular recognition. Significance: Fold plasticity influences protein evolution toward multiple function and binding promiscuity. Proteases carry out a number of crucial
... ctions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the ␤-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition. * This work was supported by grants from the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO), and the Onderzoeksraad of the Vrije Universiteit Brussel. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental methods, Tables S1 and S2, and Figs. S1-S4. The atomic coordinates and structure factors (codes 3S8K and 3S8J) have been deposited in the Protein FIGURE 4. Solution structure of PPI and the PPI-trypsin complexes. A, experimental SAXS scattering curves for free PPI and the PPI-trypsin and PPI-(trypsin) 2 complexes (from top to bottom). a.u., arbitrary units. B, distance distribution function of free PPI, PPI-trypsin, and PPI-(trypsin) 2 in cyan, light blue, and dark blue, respectively. C-E, SAXS-reconstructed pseudo-atomic structures of PPI (C), PPI-trypsin complex (D), and PPI-(trypsin) 2 complex (E). The rigid body models are superposed onto ab initio reconstructed shapes.