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J. Biol. Chem., Vol. 276, Issue 19, 16425-16431, May 11, 2001

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Crystal Structure of Activated CheY
COMPARISON WITH OTHER ACTIVATED RECEIVER DOMAINS^*

Seok-Yong Lee^§, Ho. S. Cho^¶, Jeffrey G. Pelton, Dalai Yan^¶, Edward A. Berry, and David E. Wemmer^§**

From the  Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 and the ^§ Graduate Group in Biophysics, the ^¶ Department of Plant and Microbial Biology, and the  Department of Chemistry, University of California, Berkeley, California 94720

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-Spo0A^r. 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 -strand 4 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 4/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 4/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 FixJ^r. A common feature in the active sites of BeF-CheY, P-Spo0A^r, P-FixJ^r, and phosphono-CheY is a salt bridge between Lys¹⁰⁹ N 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.

The atomic coordinates and the structure factors (code 1FQW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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