Crystal Structure of Activated CheY

Abstract

The crystal structure of BeF -activated CheY, with manganese in the magnesium binding site, was determined at 2.4-Å resolution. BeF bonds to Asp57, the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr87 and Lys109, respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF , the side chain oxygens of Asp13 and Asp57, the carbonyl oxygen of Asn59, and two water molecules. All of the active site interactions seen for BeF -CheY are also observed in P-Spo0Ar. Thus, BeF activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr106 is found buried within a hydrophobic pocket formed by β-strand β4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu89. This hydrogen bond appears to stabilize the active conformation of the β4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the β4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJr. A common feature in the active sites of BeF -CheY, P-Spo0Ar, P-FixJr, and phosphono-CheY is a salt bridge between Lys109 Nζ and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr87 and Tyr106(Thr-Tyr coupling), formation of the Lys109-PO salt bridge is directly involved in the activation of receiver domains generally.