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

Abstract 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 auto-phosphorylated 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 α4 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 intra-molecular association of the CckA DHp/Rec domains and discuss the role of a FATGUY motif, a distinguishing feature of CckARec orthologs.