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

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 ( Sv WhiD), which differs from S. coelicolor WhiD ( Sc WhiD) only at the C terminus. We observed that, like Sc WhiD and other Wbl proteins, Sv WhiD binds a [4Fe-4S] cluster that is moderately sensitive to O ₂ and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that Sv WhiD 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 Sv WhiD interacts with domain 4 of the principal sigma factor of Streptomyces , σ ^HrdB (σ ^HrdB ₄ ). Using MS, we determined the dissociation constant ( K _d ) for the Sv WhiD-σ ^HrdB ₄ 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 Sv WhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O ₂ and NO when Sv WhiD was bound to σ ^HrdB ₄ , consistent with protection of the cluster in the complex.
(© 2020 Stewart et al.)