Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin

Moh'd A. Salameh, Alexei S. Soares, Duraiswamy Navaneetham, Dipali Sinha, Peter N. Walsh, Evette S. Radisky

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Abstract

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P1 (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P2′ favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P1 and P2′ substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive.Wealso report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

Original languageEnglish (US)
Pages (from-to)36884-36896
Number of pages13
JournalJournal of Biological Chemistry
Volume285
Issue number47
DOIs
StatePublished - Nov 19 2010

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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