title: Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates.
abstract: Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and.
authors.forename: Alexandre P
authors.initials: AP
authors.lastname: Alloy
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and.
authors.forename: Olumide
authors.initials: O
authors.lastname: Kayode
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and.
authors.forename: Ruiying
authors.initials: R
authors.lastname: Wang
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hockla
authors.affiliation: the Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: Alexei S
authors.initials: AS
authors.lastname: Soares
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224 and radisky.evette@mayo.edu.
authors.forename: Evette S
authors.initials: ES
authors.lastname: Radisky
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D007611:Aprotinin / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002417:Cattle; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D051381:Rats; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics*; D014361:Trypsin Inhibitors / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D014361:Trypsin Inhibitors; D007611:Aprotinin; C040749:PRSS3 protein, human; D014357:Trypsin
keywords: crystal structure; enzyme catalysis; protease inhibitor; protein evolution; protein structure; proteolysis; serine protease; site-directed mutagenesis; substrate specificity; trypsin