A key hydrophobic patch identified in an AAA+ protein essential for its in trans inhibitory regulation

Nan Zhang, Timothy Simpson, Edward Lawton, Povilas Uzdavinys, Nicolas Joly, Patricia Burrows, Martin Buck

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA + (ATPases Associated with various cellular Activities) protein family. They are responsible for σ54-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 σ54 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.

Original languageEnglish (US)
Pages (from-to)2656-2669
Number of pages14
JournalJournal of Molecular Biology
Volume425
Issue number15
DOIs
StatePublished - Aug 9 2013

Keywords

  • PspA enhancer
  • PspF
  • binding protein
  • proteins
  • σ54

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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