Structure of the magnesium-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis

Abstract

The response regulator protein of bacterial chemotaxis, CheY, is representative of a large family of signal transduction proteins that function as phosphorylation-activated switches to regulate the activities of associated effector domains. These regulators catalyze the metal ion-dependent phosphoryl transfer and dephosphorylation reactions that control the effector activities. The crystal structures of Salmonella typhimurium CheY with and without Mg2+ bound at the active site have been determined and refined at 1.8-A resolution. While the overall structures of metal-bound and metal-free CheY are similar, significant rearrangements occur within the active site involving the three most highly conserved residues of the response regulator family. Conservation of the cluster of carboxylate side chains at the active site of response regulator domains can be rationalized in terms of their role in coordinating the catalytically essential divalent metal ion. The Mg2+ coordination geometry provides insights to the mechanism of phosphoryl transfer.

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

“…However, Mg 2ϩ binding to CheY D13K is not detectable by either resonance shifts in 19 F NMR spectra of 4-fluorophenylalanine-labeled protein or by quenching of Trp 58 fluorescence (28,34). Lack of metal ion binding to the mutant protein is consistent with the observation that in wild-type CheY, Asp 13 is directly involved in coordination of the Mg 2ϩ ion (15,16). Furthermore, D13K is phosphorylated only very slowly in the presence of the histidine protein kinase CheA and ATP (26,27), and apparently not at all in the presence of acetyl phosphate (34).…”

“…6). In the apo forms, Asp 57 and Lys 109 have strong bonding interactions, but introduction of Mg 2ϩ or Ca 2ϩ eliminates this interaction (15,16,54,56). Phosphorylation of Asp 57 is certain to rearrange the active site even further, requiring a repositioning of the Lys 109 side chain (14).…”

“…Second, both proteins might have crystallized in the active conformation. This is unlikely because the conformation observed in crystalline CheY D13K is the same as wild-type, apoCheY, both crystalline (13)(14)(15) and in solution (17)(18)(19), and the conformation of wild-type CheY has been shown to change upon Mg 2ϩ binding (15,16,18,28,42) and phosphorylation (28,29). Thus, these results strongly suggest the third possibility that the conformation of D13K observed here does not represent the activated state of CheY.…”

“…The three-dimensional structure of CheY has been solved by both x-ray crystallography (13)(14)(15)(16) and multidimensional NMR (17)(18)(19). Response regulator active sites are formed from five highly conserved residues of the response regulator superfamily (20).…”

“…Response regulator active sites are formed from five highly conserved residues of the response regulator superfamily (20). In CheY, these are Asp 12 and Asp 13 , involved in binding a Mg 2ϩ ion essential for the phosphorylation and dephosphorylation reactions (15,16,21); Asp 57 , the site of phosphorylation (22); Thr 87 , implicated in the postphosphorylation signaling events (23); and Lys 109 , also required for postphosphorylation steps in the signaling pathway (24). The structure of CheY-P has not yet been determined due to the fact that the phosphoryl group has a half-life of only a few seconds on CheY (8,9).…”