A bacterial NO-binding sensor domain evolved through acquisition of a cytochrome-derived c-type heme-binding motif

Abstract Diverse bacterial sensory domains detect intracellular and extracellular cues and relay this information to downstream signaling pathways. Per-Arnt-Sim (PAS) domains are well known to bind cofactors such as heme for intracellular sensing, whereas Cache domains - the largest family of bacterial extracytoplasmic sensor modules - are thought to function exclusively as cofactor-independent ligand-binding domains. Here, we identify and characterize a large family of Cache domains that contain a covalently bound c-type heme. Systematic sequence analysis of the DUF3365/Tll0287 family of unknown function reveals exceptional conservation of a canonical CXXCH heme-binding motif, establishing c-type heme binding as a defining feature of this domain family. Structure- and sequence-based comparisons demonstrate that, despite prior annotation as PAS-like, these domains belong to the Cache superfamily and share a conserved Cache fold augmented by a distinct structural element harboring the heme-binding motif. Heme containing Cache domains are widely distributed across bacterial phyla, with strong enrichment in Gracilicutes, and frequently occur as stand-alone modules, an atypical presentation of Cache domains. Phylogenetic analyses support insertion of a short cytochrome c–derived fragment containing the heme-binding motif into a preexisting Cache scaffold, representing a previously unrecognized mode of sensor-domain evolution. Spectral and ligand-binding analyses indicate that heme containing Cache domains fused to signal-transduction modules have properties typical of nitric oxide, but not oxygen, sensors; whereas stand-alone domains likely have redox-related roles. Our findings expand the functional repertoire of Cache domains to include covalently bound cofactors and reveal fragment-level recruitment of enzymatic motifs as a route for the emergence of bacterial sensory systems. Competing Interest Statement The authors have declared no competing interest.