Solution Structure and Dynamic Character of the Histidine-Containing Phosphotransfer Domain of Anaerobic Sensor Kinase ArcB from Escherichia coli,

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Abstract
An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions. Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPtArcB), which has a phosphorylation site, His717. The backbone dynamics were also investigated by analyses of the 15N relaxation data and amide hydrogen exchange rates. Furthermore, the protonation states of the histidine imidazole rings were characterized by means of 1H and 15N chemical shifts at various pHs. The determined solution structure of HPtArcB contains five helices and forms a four-helix bundle motif like other HPt domains. The obtained order parameters, S 2, {1H}−15N heteronuclear NOE values, and chemical exchange parameters, Rex, showed that the α-helical regions of HPtArcB are rigid on both picosecond to nanosecond and microsecond to millisecond time scales. On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D. These results suggest that HPtArcB may undergo a small conformational change in helix D upon phosphorylation. It was also shown that the imidazole ring of His717 has a pKa value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other. This is consistent with the solution structure of HPtArcB, in which the imidazole ring of His717 is exposed to the solvent.