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.
Highlights
The bacterial protein WhiD belongs to the Wbl family of ironsulfur [Fe-S] proteins present only in the actinomycetes
All previous studies have reported that Wbl proteins are monomers, we found that S. venezuelae WhiD (SvWhiD) exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension
Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, HrdB (HrdB4)
Summary
The bacterial protein WhiD belongs to the Wbl family of ironsulfur [Fe-S] proteins present only in the actinomycetes. The WhiB-like (Wbl) family of [4Fe-4S] cluster-containing proteins is found exclusively in Actinobacteria, including soil dwelling Streptomyces bacteria, which are the most abundant source of clinically important antibiotics, and Mycobacterium. The NMR structure of M. tuberculosis [4Fe-4S] WhiB1 was recently reported, the first for any Wbl protein [12], revealing a four-helix bundle with the [4Fe-4S] cluster coordinated at the interface of helices 1, 2, and 3 by the four conserved Cys residues. It was shown that WhiB1 forms a stable complex with the C-terminal part (domain 4) of SigA, the cell’s major sigma factor [12], and other M. tuberculosis Wbl proteins have been shown to interact with SigA [13, 14]. For interactions with sigma factors, the WhiB1-A4 interaction is dominated by hydrophobic interactions around the [4Fe-4S] clusterbinding pocket [15], accounting for why the cluster is essential for stability of the complex [12, 15]
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