Intermediaries in phosphotransfer: structural features discriminating hybrid histidine kinase Rec domains from response regulator homologs

In two-component systems, the information gathered by the histidine kinase (HK) is relayed to the cognate response regulator (RR). Thereby, the phosphoryl group of the autophosphorylated HK is transferred to the receiver domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec domain (Recinter) that is part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about Recinter domains and their potentially discriminating features. As a bona-fide Recinter domain, here we have studied the C-terminal Rec domain of the hybrid HK CckA (CckARec) from Caulobacter crescentus by X-ray crystallography and NMR spectroscopy. CckARec exhibits the canonical Rec-fold, though with a degenerated 4 helix, in which all active site residues are pre-arranged for phosphoryl-binding. BeF3- binding does not alter secondary structure nor the oligomeric state, indicating the absence of allosteric changes, the hall mark of RRs. Based on structural modeling and sequence co-variation analysis, we present a detailed picture for the intramolecular association of the CckA DHp/Rec domains and discuss the role of a FATGUY motif, a distinguishing feature of CckARec orthologs.