Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

Melissa Stewart, Matthew Bush, Jason Crack, Mark Buttner, Nick Le Brun

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Abstract

The bacterial protein WhiD belongs to the Wbl family of iron–sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer–dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD–σHrdB4 complex as ~0.7 μM, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.
Original languageEnglish
Pages (from-to)9752-9765
Number of pages14
JournalJournal of Biological Chemistry
Volume295
Issue number28
Early online date17 Apr 2020
DOIs
Publication statusPublished - 10 Jul 2020

Keywords

  • Streptomyces
  • Wbl proteins
  • WhiD
  • bacterial gene regulation
  • iron-sulfur cluster
  • iron-sulfur protein
  • mass spectrometry (MS)
  • metalloprotein
  • microbiology
  • nitric oxide
  • protein dimerization
  • protein-protein interaction
  • sigma factor
  • sporulation

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