Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain

Abstract

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein-protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

Figure 1

Receptor mediated signaling in bacterial chemotaxis. A family of dimeric transmembrane chemoreceptors sense changes in external levels of chemical attractants or repellents (▿) in the surrounding medium. The opposing activities of the S-adenosylmethionine-dependent methyltransferase CheR and the phosphorylation-regulated methylesterase CheB control receptor methylation levels, which in turn influence the signaling output of the chemoreceptor–histidine kinase CheA–coupling protein CheW complex. CheA autophosphorylation and subsequent transfer of the phosphoryl group to the response regulators CheY or CheB result in stimulation of effector responses: phospho-CheY controls the direction of flagellar motor rotation, whereas phospho-CheB serves to attenuate the response by hydrolysis of specific methyl glutamate residues on the receptor. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; MeOH, methanol.

Figure 2

Stereo view of a representative portion of the initial electron density map calculated after positional refinement of the molecular replacement solution overlaid with the final refined model of CheB. The electron density map was calculated to a resolution of 2.7 Å by using phases calculated from the model containing the C-terminal domain and the polyalanine model of the CheY structure in place of the CheB N-terminal domain. This model does not contain CheB residues 134–153. For the sake of clarity, only the density associated with linker residues His-138 to Thr-145 are displayed. Even at this stage the initial electron density map exhibited distinct features of side-chain density and allowed for sequence assignment for the interdomain linker. The figure was generated by using turbo (16).

Figure 3

Structure of the methylesterase CheB. (A) Ribbon representation showing the overall fold of residues 1–347 and the relative positioning of the two domains. The N-terminal regulatory domain is colored blue, the C-terminal catalytic domain is shown in green, and the linker region is colored gold. The acidic cluster of active site residues (Asp-11, Asp-12 ,and Asp-56, the site of phosphorylation) in the N-terminal domain and the catalytic triad residues (Ser-164, His-190, Asp-286) in the C-terminal domain are shown as Corey–Pauling–Koltun (CPK) models with carbon atoms colored gray, oxygen red, and nitrogen, dark blue. For consistency, all of the α-helices and β-strands are labeled analogously to the numbering schemes used previously in descriptions of CheB_c (9) and CheY (19). The β-strands within the C-terminal domain are additionally labeled by the prefix “c” (cβ1, cβ2, … ). (B) Stereo diagram of the interdomain region. The molecule is slightly rotated horizontally compared with the orientation in A. Helices and strands are shown as lines with the side chains of residues involved in the interdomain interaction represented by balls and sticks. The coloring scheme and CPK models are as indicated in A. The labels identify residues that show the greatest decrease in solvent accessibility in intact CheB as compared with that calculated for residues in the isolated domains. (C) The electrostatic potential of the molecular surface of CheB shown rotated approximately 90° about the vertical axis relative to the view in A. The view is looking toward the active site. Ser-164 sits within a funnel-like opening formed at the domain interface. In the vicinity of the active site the electrostatic potential is significantly more negative in the intact protein than in the isolated C-terminal domain, and this may have an effect on catalysis. The surface in C was calculated and displayed with the negative surfaces in red, the positive surfaces in blue, and the neutral surfaces in white by using the program grasp (20); A and B and all subsequent figures were prepared by using the program ribbons (21).

Figure 4

Structural comparison of the chemoreceptor modification enzymes. Structures of the methyltransferase CheR and the methylesterase CheB were aligned on the basis of similarity of their C-terminal domains by using a structural homology search in dali (34). For both molecules, ribbon diagrams depict the N-terminal domains in blue, linker regions in gold, and C-terminal domains in green. The molecule of S-adenosylhomocysteine (SAH) in CheR and the methylesterase active site residues [Ser-164 (S), His-190 (H), and Asp-286(D)] in CheB are shown as CPK models. The double-headed arrow points toward the active sites and the receptor interaction openings. Functionally antagonistic CheB and CheR contain active sites on opposite faces of the structurally homologous central β-sheets.

Figure 5

CPK model of the N-terminal domain of CheB, showing surfaces that are involved in protein–protein interactions among the response regulators CheB, CheY, and NarL. Residues involved in interaction with the C-terminal domain of CheB are colored yellow. Residues that have been implicated in protein–protein interactions in other response regulators are shown with colored mesh: red for corresponding residues in CheY that are thought to be involved in interaction with the P2 domain of the histidine kinase CheA (39); green for corresponding residues in NarL that interact with its C-terminal DNA-binding domain (25).