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. We also 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.
authors.affiliation: Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, Florida 32224, USA.
authors.forename: Moh'd A
authors.initials: MA
authors.lastname: Salameh
authors.forename: Alexei S
authors.initials: AS
authors.lastname: Soares
authors.forename: Duraiswamy
authors.initials: D
authors.lastname: Navaneetham
authors.forename: Dipali
authors.initials: D
authors.lastname: Sinha
authors.forename: Peter N
authors.initials: PN
authors.lastname: Walsh
authors.forename: Evette S
authors.initials: ES
authors.lastname: Radisky
mesh_terms: D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism*
chemical_list: D016564:Amyloid beta-Protein Precursor; D011994:Recombinant Proteins; D007611:Aprotinin; C040749:PRSS3 protein, human; D014357:Trypsin