title: Determination of the self-association residues within a homomeric and a heteromeric AAA+ enhancer binding protein.
abstract: The σ(54)-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ(54)-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars. Unlike other bEBPs that usually act as homohexamers, HrpR and HrpS operate as a highly co-dependent heterohexameric complex. To understand the organization of the HrpRS complex and the HrpR and HrpS strict co-dependence, we have analyzed the interface between subunits using the random and directed mutagenesis and available crystal structures of several closely related bEBPs. We identified key residues required for the self-association of HrpR (D32, E202 and K235) with HrpS (D32, E200 and K233), showed that the HrpR D32 and HrpS K233 residues form interacting pairs directly involved in an HrpR-HrpS association and that the change in side-chain length at position 233 in HrpS affects self-association and interaction with the HrpR and demonstrated that the HrpS D32, E200 and K233 are not involved in negative regulation imposed by HrpV. We established that the equivalent residues K30, E200 and E234 in a homo-oligomeric bEBP, PspF, are required for the subunit communication and formation of an oligomeric lock that cooperates with the ATP γ-phosphate sensing PspF residue R227, providing insights into their roles in the heteromeric HrpRS co-complex.
authors.affiliation: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: Lawton
authors.affiliation: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Milija
authors.initials: M
authors.lastname: Jovanovic
authors.affiliation: Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Batiment Buffon, 15 rue Helene Brion, 75205 Paris Cedex 13, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Joly
authors.affiliation: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Waite
authors.affiliation: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, UK.
authors.forename: Baojun
authors.initials: B
authors.lastname: Wang
authors.affiliation: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Patricia
authors.initials: P
authors.lastname: Burrows
authors.affiliation: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK. Electronic address: m.buck@imperial.ac.uk.
authors.forename: Martin
authors.initials: M
authors.lastname: Buck
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D044224:Pseudomonas syringae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C085856:HrpR protein, Pseudomonas syringae; D021122:Protein Subunits; D011994:Recombinant Proteins; D014157:Transcription Factors; C060789:hrpS protein, Pseudomonas syringae; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases
keywords: Abbreviations; BACTH; HrpR; HrpS; Pseudomonas syringae; PspF; RNA polymerase; RNAP; bEBP; bacterial enhancer binding protein; bacterial two-hybrid; σ(54)