Beltran, D. G.; Wan, T.; Seravalli, J.; Horova, M.; Jones, C.; Li, S.; Ma, P.; Becker, D.; Mower, J. P.; Yao, Q.; Pan, Y.; Yu, H.; Steyn, A. J.; Zhang, L.

WhiB-like (Wbl) family proteins are a group of small iron-sulfur ([4Fe-4S]) cluster-containing transcription factors and are uniquely found in Actinobacteria and actinobacteriophages. They play pivotal roles in diverse biological processes, including cell development, redox stress response and antibiotic resistance. However, the lack of a canonical DNA binding motif in most Wbl proteins has long obscured the mechanistic understanding of how Wbl family proteins regulate gene expression. Previous studies have shown several members of Wbl family proteins from Mycobacterium tuberculosis (Mtb) and Streptomyces interact with the conserved region 4 of the primary sigma factor ({sigma}A4) in RNA polymerase holoenzyme for transcriptional regulation. The molecular interface between Wbls and {sigma}A4 is unexpectedly dominated by hydrophobic interactions featuring a patch of conserved aromatic residues within the iron-sulfur cluster binding pocket. In this study, we present structural, molecular and biochemical evidence demonstrating that all Mtb Wbls bind to the same site on {sigma}A using a patch of conserved aromatic residues (hereby referred to as the aromatic patch) within the iron-sulfur cluster binding pocket. Moreover, our bioinformatic analyses and structural modeling of representative Wbl family proteins reveal that this aromatic patch is a conserved structural motif across the Wbl family. Our findings provide compelling evidence that Wbl proteins regulate gene expression via a shared interaction interface with {sigma}A4 and uncover a complex evolutionary relationship of Wbls between actinobacteria and their associated phages.