Production, characterization, and assessment of a stable analog of the response regulator CheY-phosphate from Thermotoga maritima

Abstract

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with a dissociation constant of ∼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate.

Keywords: chemotaxis; cysteine modification; phosphate-analog; two-component system.

Figure 1

Activation of Tm CheY. CheY is phosphorylated in vivo by CheA kinase to generate activated CheY‐P. CheY‐P is rapidly dephosphorylated (half‐life of ∼20 s) by the CXY family of phosphatases (CheC/CheX/FliY) and through water hydrolysis. To enable biochemical study of activated Tm CheY, a nonhydrolyzable analog has been generated by alkylating D54C/C81A CheY with phosphonomethyltriflate. (PDB 4QYW).

Figure 2

Mass spectrometric analysis of (a) intact CheY‐pP and (b) Cys54 tryptic peptide. The mass spectra of intact CheY‐pP (M = 13548 Da) indicates an increase in mass of 94 Da relative to CheY, corresponding to addition of the phosphonomethyl group. A minor oxidized species is also observed at 13564 Da. The CID tandem mass spectra of the quadruply charged tryptic peptide localizes the phosphonomethyl group to the desired cysteine residue. Mass peaks correspond to the b and y fragment ions shown above.

Figure 3

(a) The phosphonomethyl group (shown in pink) is modeled in two different orientations approximately 120° apart in the active site of Tm D54C/C81A CheY‐pP (PDB 4QYW). (b) 2Fo–Fc electron density map (blue contours at 1σ) and Fo–Fc map (green contours at +3σ; red contours at −3σ) depicting the modified Cys54 in one of two modeled conformations. The phosphonomethylated Cys54 residue is omitted from Fc in both maps. In one conformation, the phosphonomethyl group interacts with the amino‐containing side chain of Gln86; in the other the phosphonomethyl group is positioned ∼3.2 Å from Lys104, forming an important ionic interaction (not shown in this depiction).

Figure 4

Direct8 binding between FITC‐labeled FliM_N peptide and Tm CheY‐pP observed using fluorescence polarization. Dissociation constants were measured by monitoring changes in fluorescence polarization as increasing concentrations of CheY protein were added to 25 nM fluorescein‐labeled FliM_N peptide. The experiments were performed using a buffer system containing 50 mM MOPS pH 7.2, 20 mM MgCl₂.