title: X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.
abstract: Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.
authors.affiliation: Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Kerstin B
authors.initials: KB
authors.lastname: Rohr
authors.forename: Trevor
authors.initials: T
authors.lastname: Selwood
authors.forename: Ulf
authors.initials: U
authors.lastname: Marquardt
authors.forename: Robert
authors.initials: R
authors.lastname: Huber
authors.forename: Norman M
authors.initials: NM
authors.lastname: Schechter
authors.forename: Wolfram
authors.initials: W
authors.lastname: Bode
authors.forename: Manuel E
authors.initials: ME
authors.lastname: Than
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053802:Tryptases
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D007976:Leupeptins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053802:Tryptases; C032854:leupeptin