Determination of the self-association residues within a homomeric and a heteromeric AAA plus enhancer binding protein

Edward Lawton, Milija Jovanovic, Nicolas Joly, Christopher Waite, Nan Zhang, Baojun Wang, Patricia Burrows, Martin Buck*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


The sigma(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 sigma(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 gamma-phosphate sensing PspF residue R227, providing insights into their roles in the, heteromeric HrpRS co-complex. (C) 2014 Published by Elsevier Ltd.

Original languageEnglish
Pages (from-to)1692-1710
JournalJournal of Molecular Biology
Issue number8
Publication statusPublished - 13 Jan 2014


  • sigma 54
  • PspF
  • HrpR
  • HrpS
  • pseudomonas syringae
  • escherichia-coli
  • transcriptional activation
  • RNA-polymerase
  • in-vivo
  • SIGMA(54)
  • PSPF
  • mechanism
  • promoter
  • HRP


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