An NO-binding Cache domain receptor interacts with a Ser/Thr kinase through a conserved HAMP domain interaction
Cache_heme domains are a family of bacterial heme c proteins that combine a conserved Cache fold with an -helical insertion containing a heme-binding CXXCH motif. We characterize an unusual Cache_heme-HAMP (CHH) receptor from Pseudomonas azotoformans with a periplasmic NO-binding Cache_heme domain, transmembrane region, and a cytoplasmic HAMP domain uncoupled from intrinsic enzymatic output. Genomic analysis reveals that CHH receptors are frequently co-localized with genes encoding Ser/Thr kinases (STPKs) that possess a catalytic domain similar to PknB, but lack peptidoglycan-binding PASTA domains. Using biochemical assays, biolayer interferometry, SEC-MALS, and cryo-electron microscopy, we show that the P. azotoformans CHH receptor binds its operon-associated kinase with nanomolar affinity via conserved C-terminal repeat modules in the kinase composed of two pseudo-symmetric -helical bundles. Cryo-EM structures demonstrate that this interaction orders the otherwise flexible HAMP domain, with the repeated helical domains from the kinase contacting each HAMP subunit symmetrically. Kinase-receptor binding is ATP-independent, but kinase phosphorylation of the receptor at a specific HAMP threonine residue is substantially enhanced when NO binds to the heme c sensor domain. Thus, NO-induced conformational changes that transverse the membrane either modulate substrate accessibility in the HAMP or alter kinase activity directly. These findings potentially define a mode of bacterial NO signaling in which a periplasmic heme c receptor couples ligand sensing to cytoplasmic phosphorylation via a physically associated Ser/Thr kinase, a mechanism distinct from that of cytoplasmic NO sensors. The conserved nature of the kinase recognition motif suggests broader relevance of CHH-STPK interactions across proteobacteria.