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Title: Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

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 P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} 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 P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report themore » crystal structure of the mesotrypsin {center_dot} 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
DOE - OFFICE OF SCIENCE
OSTI Identifier:
1014336
Report Number(s):
BNL-94522-2010-JA
Journal ID: ISSN 0021-9258; JBCHA3; R&D Project: BO-070; KP1605010; TRN: US201111%%289
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 285; Journal Issue: 47; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AFFINITY; CATTLE; CLEAVAGE; CRYSTAL STRUCTURE; FLEXIBILITY; FUNCTIONALS; INACTIVATION; PRECURSOR; PROTEINS; PROTEOLYSIS; RESIDUES; STABILITY; THERMODYNAMICS; TRYPSIN; VULNERABILITY

Citation Formats

Salameh, M A, Soares, A, Navaneetham, D, Sinha, D, Walsh, P N, and Radisky, E S. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin. United States: N. p., 2010. Web. doi:10.1074/jbc.M110.171348.
Salameh, M A, Soares, A, Navaneetham, D, Sinha, D, Walsh, P N, & Radisky, E S. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin. United States. https://doi.org/10.1074/jbc.M110.171348
Salameh, M A, Soares, A, Navaneetham, D, Sinha, D, Walsh, P N, and Radisky, E S. 2010. "Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin". United States. https://doi.org/10.1074/jbc.M110.171348.
@article{osti_1014336,
title = {Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin},
author = {Salameh, M A and Soares, A and Navaneetham, D and Sinha, D and Walsh, P N and Radisky, E S},
abstractNote = {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 P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} 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 P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin {center_dot} 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.},
doi = {10.1074/jbc.M110.171348},
url = {https://www.osti.gov/biblio/1014336}, journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 47,
volume = 285,
place = {United States},
year = {Fri Nov 19 00:00:00 EST 2010},
month = {Fri Nov 19 00:00:00 EST 2010}
}