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

Abstract

<jats:p>The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In <jats:italic>Streptomyces coelicolor</jats:italic>, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic> experiments with WhiD from <jats:italic>Streptomyces venezuelae</jats:italic> (<jats:italic>Sv</jats:italic>WhiD), which differs from <jats:italic>S. coelicolor</jats:italic> WhiD (<jats:italic>Sc</jats:italic>WhiD) only at the C terminus. We observed that, like <jats:italic>Sc</jats:italic>WhiD and other Wbl proteins, <jats:italic>Sv</jats:italic>WhiD binds a [4Fe-4S] cluster that is moderately sensitive to O<jats:sub>2</jats:sub> and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that <jats:italic>Sv</jats:italic>WhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of <jats:italic>Mycobacterium tuberculosis</jats:italic> are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that <jats:italic>Sv</jats:italic>WhiD interacts with domain 4 of the principal sigma factor of <jats:italic>Streptomyces</jats:italic>, σ<jats:sup>HrdB</jats:sup> (σ<jats:sup>HrdB</jats:sup><jats:sub>4</jats:sub>). Using MS, we determined the dissociation constant (<jats:italic>K<jats:sub>d</jats:sub></jats:italic>) for the <jats:italic>Sv</jats:italic>WhiD-σ<jats:sup>HrdB</jats:sup><jats:sub>4</jats:sub> complex as ∼0.7 μ<jats:sc>m</jats:sc>, 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 <jats:italic>Sv</jats:italic>WhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O<jats:sub>2</jats:sub> and NO when <jats:italic>Sv</jats:italic>WhiD was bound to σ<jats:sup>HrdB</jats:sup><jats:sub>4</jats:sub>, consistent with protection of the cluster in the complex.</jats:p>

Characterization of a [4Fe-4S]-dependent LarE sulfur insertase that facilitates nickel-pincer nucleotide cofactor biosynthesis in Thermotoga maritima

“…Anoxic samples of His 6 -tagged LarE Tm were purified from 0.3 l of aerobically grown E. coli cultures supplemented with 200 μM ammonium ferric citrate and 25 μM L-methionine ( 37 ) within an anaerobic chamber (Coy Laboratory Products) that contained an atmosphere of 95% N 2 plus 5% H 2 (<2 ppm [O 2 ]) and a palladium catalyst. The cells were lysed by addition of 0.2 to 0.3 mg/ml lysozyme, followed by incubation at 37 ⁰C for 1 h. Cell-free lysates were obtained by centrifugation at 13,800 g for 1 h at RT.…”

Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis

“…Domain 4 of s A is a highly conserved motif among the s 70 -family primary sigma factors in bacteria, which recognizes the À35 element and interacts with many transcription factors for regulating gene expression (Feng et al, 2016;Gomez et al, 1998;Lee et al, 2012). Several other Wbl proteins also regulate gene expression via interaction with s A 4 , indicating a shared mode of action by Wbl proteins (Feng et al, 2016;Kudhair et al, 2017;Stewart et al, 2020;Steyn et al, 2002;Wan et al, 2020).…”

Sensing mechanisms of iron–sulfur cluster regulatory proteins elucidated using native mass spectrometry

“…Native MS studies of both WhiB1 from M. tuberculosis and WhiD from S. venezuelae have demonstrated 1:1 complex formation that is dependent on the [4Fe-4S] cluster. These complexes were either insensitive (WhiB1) or less sensitive (WhiD) to O 2 , but reacted rapidly with NO, leading to dissociation of the complex, 46,103 supporting the idea that these proteins function in sensing NO. resolved native MS studies of reactions of Fe-S cluster cofactors within a protein framework.…”