title: Mapping a ketosynthase:acyl carrier protein binding interface via unnatural amino acid-mediated photo-cross-linking.
abstract: Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.
authors.affiliation: Department of Chemistry, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Zhixia
authors.initials: Z
authors.lastname: Ye
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Williams
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D055668:Photochemical Processes; D054730:Protein Interaction Domains and Motifs; D016930:Saccharopolyspora / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D000123:Acetyltransferases; C557137:fabF protein, E coli; D054889:Fatty Acid Synthase, Type II