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

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 1 (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P 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 P 1 and P 2′ 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

Fingerprint

Amyloid beta-Protein Precursor
Protease Inhibitors
Aprotinin
Scaffolds
Proteolysis
Sequence Homology
Thermodynamics
Trypsin
Proteins
Substitution reactions
Crystal structure
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin. / Salameh, Moh'd A.; Soares, Alexei S.; Navaneetham, Duraiswamy; Sinha, Dipali; Walsh, Peter N.; Radisky, Evette S.

In: Journal of Biological Chemistry, Vol. 285, No. 47, 19.11.2010, p. 36884-36896.

Research output: Contribution to journalArticle

Salameh, Moh'd A. ; Soares, Alexei S. ; Navaneetham, Duraiswamy ; Sinha, Dipali ; Walsh, Peter N. ; Radisky, Evette S. / Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 47. pp. 36884-36896.
@article{e2cbc3bd719d4432b5777a7443041c6a,
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 1 (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P 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 P 1 and P 2′ 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.",
author = "Salameh, {Moh'd A.} and Soares, {Alexei S.} and Duraiswamy Navaneetham and Dipali Sinha and Walsh, {Peter N.} and Radisky, {Evette S}",
year = "2010",
month = "11",
day = "19",
doi = "10.1074/jbc.M110.171348",
language = "English (US)",
volume = "285",
pages = "36884--36896",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "47",

}

TY - JOUR

T1 - Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin

AU - Salameh, Moh'd A.

AU - Soares, Alexei S.

AU - Navaneetham, Duraiswamy

AU - Sinha, Dipali

AU - Walsh, Peter N.

AU - Radisky, Evette S

PY - 2010/11/19

Y1 - 2010/11/19

N2 - 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 1 (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P 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 P 1 and P 2′ 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.

AB - 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 1 (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P 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 P 1 and P 2′ 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.

UR - http://www.scopus.com/inward/record.url?scp=78449246593&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78449246593&partnerID=8YFLogxK

U2 - 10.1074/jbc.M110.171348

DO - 10.1074/jbc.M110.171348

M3 - Article

VL - 285

SP - 36884

EP - 36896

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 47

ER -