A Shared Anchor on Primary Sigma Factor SigA by the WhiB‐Like Proteins

WhiB-like (Wbl) proteins are a unique group of iron-sulfur cluster ([4Fe4S])-containing transcription factors exclusive to actinobacteria. They play crucial roles in the virulence, survival, and propagation of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Seven Wbl proteins (WhiB1-WhiB7) are found in Mtbwith diverse regulatory roles. All Mtb Wbl proteins except for WhiB5 have been shown to interact with the conserved region 4 of the primary sigma factor (SigAr4) in RNA polymerase holoenzyme. Our recent structural and biochemical analyses indicate that two Wbl proteins, WhiB1 and WhiB7, bind to the same site on SigAr4 unexpectedly through tight hydrophobic interactions involving several conserved aromatic residues of the Wbl proteins and His516 of SigAr4. Substitution of these aromatic residues in either WhiB1 or WhiB7 at the molecular interface abolishes their binding to SigAr4. Here, we hypothesize that all Mtb Wbl proteins share a similar molecular interface when in complex with SigAr4, based on the primary sequence analysis and structural modeling. Using the site-directed mutagenesis and the in vitro protein-protein interaction assays, we confirm that all Mtb Wbl proteins, including WhiB5, bind to the same site on SigA centered on His516, and the conserved aromatic residues within the Fe-S cluster binding pocket are required for SigA binding. The results from this study will offer insights into how the Wbl proteins are utilized in Mtb to orchestra gene expression in response to environmental cues in the host.