Aromatic Patch in WhiB-Like Transcription Factors Facilitates Primary Sigma Factor Interaction in .

Unlabelled: 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 ( ) and interact with the conserved region 4 of the primary sigma factor (σ ) in RNA polymerase holoenzyme for transcriptional regulation. The molecular interface between Wbls and σ 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 Wbls bind to the same site on σ 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 σ and uncover a complex evolutionary relationship of Wbls between actinobacteria and their associated phages.

Significance: This work addresses a critical knowledge gap in understanding how WhiB-like (Wbl) family proteins regulate gene expression and confer virulence and antibiotic resistance in . WhiB-like proteins are widely distributed in Actinobacteria, the phylum that includes several deadly human pathogens and important antibiotic producers. By identifying the previously unrecognized structural motif that is highly conserved in the Wbl family and mediates Wbl interaction with the core transcriptional machinery, this work advances our understanding of the evolution, function and underlying mechanism of transcriptional regulation by this unique family of transcription factors in Actinobacteria and associated phages. These insights will be instrumental in developing new strategies to combat tuberculosis and other diseases caused by related pathogens.