Solution Structures of the Inactive and BeF3-activated Response Regulator CheY2

The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2. CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA. Here, we report the NMR solution structures of the Mg2+-complex of inactive CheY2, and of activated CheY2-BeF3, a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively. The 14 kDa CheY2 protein exhibits a characteristic open (α/β)5 conformation. Modification of CheY2 by BeF3− leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58. In BeF3-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF3, similar to BeF3-activated CheY from Escherichia coli. In contrast to E. coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface. Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of α4 towards β5 is stabilised in S. meliloti. The resulting, activation-induced, compact α4-β5-α5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control. We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S. meliloti.