Abstract
Proteases carry out a number of crucial functions 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 β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.
Highlights
The Kunitz-soybean trypsin inhibitor (STI) family is a paradigm of protease-inhibitor interaction in particular and protein-protein recognition in general
Despite the abundance of data obtained from x-ray crystallography for free inhibitors and complexes with serine proteases [7,8,9, 12,13,14], very little is known on the specifics of multiple target recognition by these Janus-type proteins with only one structure in the Protein Data Bank of a Kunitz-STI inhibitor bound to two protease molecules [7]
Overall Structure of papaya protease inhibitor (PPI)—A BLAST search shows that PPI is a member of the miraculin family of taste-modifying proteins, which are active against serine proteases
Summary
The Kunitz-STI family is a paradigm of protease-inhibitor interaction in particular and protein-protein recognition in general. The Kunitz-STI family belongs to the -trefoil fold superfamily, which displays an extremely high plasticity regarding their interacting partners [5] The latter involves protein, DNA, and carbohydrate recognition, and some of them are even enzymes. Despite the abundance of data obtained from x-ray crystallography for free inhibitors and complexes with serine proteases [7,8,9, 12,13,14], very little is known on the specifics of multiple target recognition by these Janus-type proteins with only one structure in the Protein Data Bank of a Kunitz-STI inhibitor bound to two protease molecules [7]. Plasticity of the -Trefoil Fold in Molecular Recognition papaya It has a high stability and is resistant to proteolysis by proteases from different families [15]. We discuss how the potential of the -trefoil fold to accept surface mutations influences protein evolution toward multiple function and multiple target recognition
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