Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom.
J Biol Chem. 2020 Jul 10;295(28):9752-9765. doi: 10.1074/jbc.RA120.012708. Epub 2020 Apr 16.
The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In , it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from and experiments with WhiD from (WhiD), which differs from WhiD (WhiD) only at the C terminus. We observed that, like WhiD and other Wbl proteins, WhiD binds a [4Fe-4S] cluster that is moderately sensitive to O and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that WhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that WhiD interacts with domain 4 of the principal sigma factor of , σ (σ). Using MS, we determined the dissociation constant ( ) for the WhiD-σ 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 WhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O and NO when WhiD was bound to σ, consistent with protection of the cluster in the complex.
细菌蛋白 WhiD 属于铁硫（Fe-S）蛋白家族的 Wbl 家族，仅存在于放线菌中。在那里，它是孢子形成后期所必需的，但它的具体功能尚不清楚。在这里，我们报告了来自 （WhiD）的 WhiD 的 和 实验结果，它与 WhiD（WhiD）仅在 C 末端不同。我们观察到，与 WhiD 和其他 Wbl 蛋白一样，WhiD 结合一个 [4Fe-4S]簇，该簇对 O 中度敏感，对一氧化氮（NO）高度敏感。然而，尽管之前的所有研究都报道 Wbl 蛋白是单体，但我们发现 WhiD 存在单体-二聚体平衡，这与其不寻常的 C 末端延伸有关。 的几种 Wbl 蛋白已知与主要 sigma 因子 SigA 相互作用。使用细菌双杂交、凝胶过滤和 MS 分析，我们证明 WhiD 与 的主要 sigma 因子 σ（σ）的结构域 4 相互作用。使用 MS，我们确定了 WhiD-σ 复合物的离解常数（）为 ∼0.7 μm，这与相对紧密的结合相互作用一致。我们发现复合物的形成依赖于簇，并且与 NO 的反应完全在每个簇 8-10 个 NO 分子的情况下发生，导致分离成单独的蛋白质。当 WhiD 与 σ 结合时，WhiD 的 [4Fe-4S]簇对与 O 和 NO 的反应的敏感性显著降低，这与簇在复合物中的保护一致。
