title: A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase.
abstract: Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a M(r) of 43,000. In a previous paper (Occhipinti et al., Biophys J 2003; 85:1165-1175), we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the crosslink. More notably, we also identified a protease-resistant core consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. On the basis of the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380, and Leu376. Although the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, DeltaG++, DeltaH++, and DeltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sommaruga
authors.forename: Antonella
authors.initials: A
authors.lastname: De Palma
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Mauri
authors.forename: Manuela
authors.initials: M
authors.lastname: Trisciani
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Basilico
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Martelli
authors.forename: Rita
authors.initials: R
authors.lastname: Casadio
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
authors.forename: Emanuela
authors.initials: E
authors.lastname: Occhipinti
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002268:Carboxypeptidases / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010434:Pepsin A / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D012694:Serine / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013816:Thermodynamics; D014357:Trypsin / Q000494:pharmacology
chemical_list: D004220:Disulfides; D021122:Protein Subunits; D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine; D002268:Carboxypeptidases; D014357:Trypsin; D010434:Pepsin A; D003545:Cysteine