title: Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases.
abstract: Nonribosomal peptide synthetases are large, multi-domain enzymes that produce peptide molecules with important biological activity such as antibiotic, antiviral, anti-tumor, siderophore and immunosuppressant action. The adenylation (A) domain catalyzes two reactions in the biosynthetic pathway. In the first reaction, it activates the substrate amino acid by adenylation and in the second reaction it transfers the amino acid onto the phosphopantetheine arm of the adjacent peptide carrier protein (PCP) domain. The conformation of the A domain differs significantly depending on which of these two reactions it is catalyzing. Recently, several structures of A-PCP di-domains have been solved using mechanism-based inhibitors to trap the PCP domain in the A domain active site. Here, we present an alternative strategy to stall the A-PCP di-domain, by engineering a disulfide bond between the native amino acid substrate and the A domain. Size exclusion studies showed a significant shift in apparent size when the mutant A-PCP was provided with cross-linking reagents, and this shift was reversible in the presence of high concentrations of reducing agent. The cross-linked protein crystallized readily in several of the conditions screened and the best crystals diffracted to ≈8 Å.
authors.affiliation: Department of Biochemistry, McGill University, Montréal, QC, Canada H3G 0B1.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Tarry
authors.affiliation: Department of Biochemistry, McGill University, Montréal, QC, Canada H3G 0B1 Groupe de Recherche Axé sur la Structure des Protéines (GRASP), McGill University, Montréal, QC, Canada H3G 0B1 martin.schmeing@mcgill.ca.
authors.forename: T Martin
authors.initials: TM
authors.lastname: Schmeing
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D010408:Penicillium chrysogenum / Q000201:enzymology; D010453:Peptide Synthases / Q000737:chemistry*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary
chemical_list: D004220:Disulfides; D005656:Fungal Proteins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: crystallization; disulfide cross-link; nonribosomal peptide synthetase; protein engineering; thioesterification