Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for In vitro metalloregulation in the initiation of sporulation

D. J. Kojetin, R. J. Thompson, L. M. Benson, S. Naylor, J. Waterman, Keith Davies, Charles H. Opperman, Keith Stephenson, J. A. Hoch, J. Cavanagh

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and mu ESI-MS. NMR studies show that the divalent metals Ca2+, Mg2+ and Mn2+ primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu2+ show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the beta 4-alpha 4 loop and the beta 5/alpha 5 interface, particularly around and including H101. mu ESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu2+, multiple metal-bound species are observed. Subsequent experiments reveal that Mg2+ supports phosphotransfer between KinA and Spo0F, while Cu2+ fails to support KinA phosphotransfer. Additionally, the presence of Cu2+ at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.

Original languageEnglish
Pages (from-to)449-466
Number of pages18
JournalBiometals
Volume18
Issue number5
DOIs
Publication statusPublished - Oct 2005

Keywords

  • metal binding
  • NMR spectroscopy
  • response regulator
  • sporulation
  • two-component signal transduction
  • HIGH-RESOLUTION NMR
  • MOLECULAR RECOGNITION
  • SIGNAL-TRANSDUCTION
  • PHOSPHORELAY
  • PROTEINS
  • SPOOF
  • PHOSPHOTRANSFER
  • MEGATERIUM
  • REVEALS
  • PHOSPHORYLATION

Fingerprint

Dive into the research topics of 'Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for In vitro metalloregulation in the initiation of sporulation'. Together they form a unique fingerprint.

Cite this