title: A key hydrophobic patch identified in an AAA⁺ protein essential for its in trans inhibitory regulation.
abstract: Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA(+) (ATPases Associated with various cellular Activities) protein family. They are responsible for σ(54)-dependent transcription activation during infection and function under many stressful growth conditions. The majority of bEBPs are regulated in their formation of ring-shaped hexameric self-assemblies via an amino-terminal domain through its phosphorylation or ligand binding. In contrast, the Escherichia coli phage shock protein F (PspF) is negatively regulated in trans by phage shock protein A (PspA). Up to six PspA subunits suppress PspF hexamer action. Here, we present biochemical evidence that PspA engages across the side of a PspF hexameric ring. We identify three key binding determinants located in a surface-exposed 'W56 loop' of PspF, which form a tightly packed hydrophobic cluster, the 'YLW' patch. We demonstrate the profound impact of the PspF W56 loop residues on ATP hydrolysis, the σ(54) binding loop 1, and the self-association interface. We infer from single-chain studies that for complete PspF inhibition to occur, more than three PspA subunits need to bind a PspF hexamer with at least two binding to adjacent PspF subunits. By structural modelling, we propose that PspA binds to PspF via its first two helical domains. After PspF binding-induced conformational changes, PspA may then share structural similarities with a bEBP regulatory domain.
authors: Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, Exhibition Road, London SW7 2AZ, UK. nan.zhang@imperial.ac.uk
authors: Nan
authors: N
authors: Zhang
authors: Timothy
authors: T
authors: Simpson
authors: Edward
authors: E
authors: Lawton
authors: Povilas
authors: P
authors: Uzdavinys
authors: Nicolas
authors: N
authors: Joly
authors: Patricia
authors: P
authors: Burrows
authors: Martin
authors: M
authors: Buck
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008954:Models, Biological; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D051877:RNA Polymerase Sigma 54 / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C098954:PspF protein, E coli; D015534:Trans-Activators; C069816:phage shock protein, Bacteria; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54
keywords: AAA(+); AAA(+) proteins; ATPases Associated with various cellular Activities; EDTA; PDB; Protein Data Bank; PspA; PspF; RNA polymerase; RNAP; WT; bEBP; bacterial enhancer binding protein; enhancer binding protein; ethylenediaminetetraacetic acid; phage shock protein A; phage shock protein F; wild type; σ54
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