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. 1993 Jun 25;268(18):13081-8.

Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering

S K Drake  1 , R B Bourret, L A Luck, M I Simon, J J Falke
Affiliations

Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering

S K Drake et al. J Biol Chem. .

Abstract

CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation. 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper. Together these results may give insight into the activation of other prokaryotic response regulators.

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Figures

Fig. 1
Fig. 1. Structure of CheY (12)
The space-filling side chains highlight the activation site containing the phosphorylation (Asp57), the proposed salt bridge switch (Asp57-Lys109) and the carboxylate cluster Mg(II) binding site (Asp12, Asp13, Asp57). Also highlighted are the six phenylalanine positions utilized as fluorine labeling sites to monitor the following regions: the vicinity of Lys109 (Phe111), the carboxylate cluster (Phe14), and the protein framework distant from the activation site (Phe8, Phe30, Phe53, and Phe124).
Fig. 2
Fig. 2. 470-MHz NMR spectrum of 4F-Phe-labeled CheY
The two indicated spectra were obtained from different 4F-Phe CheY preparations, demonstrating the reproducibility of the fluorine chemical shifts. Six well resolved resonances are observed. Sample parameters were: 2 mM CheY, 2 mM MgCl2, 50 mM KCl, 50 mM NaCl, 50 mM Tris-HCl, pH 7.0, 10% D20, 50 μM 5F-Trp as internal frequency standard, 25 °C. Assignments were made as in Fig. 3.
Fig. 3
Fig. 3. 19F NMR resonance assignments
Summarized are the spectra of the engineered mutants used to assign the 4F-Phe resonances, either by the direct replacement method (F14Y), or by the nudge method (the others), as described in the text. Spectra of engineered proteins (bold lines) are superimposed on the spectrum of the wild-type protein (fine line). The wild-type resonance assigned by each mutation is indicated by the arrow. Sample parameters were as described in the legend to Fig. 2.
Fig. 4
Fig. 4. Effect of paramagnetic metal ions on the 19F NMR spectrum of 4F-Phe-labeled CheY
Each spectral pair consists of one spectrum obtained prior to the addition of paramagnetic probe (fine line), and a second spectrum illustrating the effects of the paramagnet (bold line). Mn(II) ion binding to the carboxylate cluster is observed to selectively broaden two resonances near the carboxylate cluster divalent cation binding site (upper, arrows), while the aqueous probe Gd(III)·EDTA broadens the two overlapping resonances exposed to solvent (lower, arrow). Sample parameters were as described in the legend to Fig. 2, except MgCl2 was omitted and additional divalent-free KCl was substituted for the NaCl.
Fig. 5
Fig. 5. Effect of group IIa metal ions on the 19F NMR spectrum of 4F-Phe-labeled CheY
Each spectral pair includes the spectrum of CheY saturated with a 20 mM concentration of the appropriate divalent metal ion (bold line), overlayed on the spectrum obtained in the absence of divalent ions (fine line). Arrows indicate the resonances exhibiting the largest divalent-induced frequency shifts. Sample parameters were as described in the legend to Fig. 4.
Fig. 6
Fig. 6. Effect of phosphorylation on the 19F NMR spectrum of 4F-Phe-labeled CheY
Shown is the spectrum of 4F-Phe CheY before the addition of acetyl phosphate as a phosphodonor (0 min), then as the reaction proceeds a mixture of phospho-CheY and CheY appears (10 min), and finally the phospho-CheY is fully hydrolyzed and the CheY population returns to its unphosphorylated state (50 min). Reaction conditions were 7 mM CheY, 25 mM MgCl2, 50 mM KCl, 50 mM NaCl, 50 mM PIPES/NaOH, pH 7.0, 10% D2O, and 150μM 5F-Trp. The reaction was initiated by addition of 200 mM acetyl phosphate, pH 7.0. The indicated assignment for 4F-Phe14 was revealed by the spectrum of the phosphorylated CheYF14Y mutant; other phospho-CheY assignments assumed the simplest pattern of shifts (see also Fig. 8).
Fig. 7
Fig. 7. Time course of the acetyl phosphate hydrolysis reaction as studied by (A), 19F NMR, and (B), 31P NMR
A, 19F NMR followed the formation and decay of phospho-CheY by integrating the resonances that were adequately frequency shifted by phosphorylation (4F-Phe8, 4F-Phe53, 4F-Phe124 + 4F-Phe30). After normalization, the integrals of these resonances were averaged to yield the indicated curve. Also shown are the corresponding curves of several key mutants. The CheYK109R mutant has previously been shown (56) to dephosphorylate atan abnormally slow rate (upper curve). The CheYD57A and CheYK109Q mutants are negative controls which failed to be phosphorylated (lower curves). B, 31P NMR enabled simultaneous monitoring of the phosphodonor and final phospho-product generated by the reaction: acetyl phosphate (−2.2 ppm) and inorganic phosphate (+1.3 ppm), respectively. The relative amount of each component was quantitated by integration of its associated resonance, and plotted as shown against time. Reaction conditions were as described in the legend to Fig. 6.
Fig. 8
Fig. 8. Comparing the 19F NMR spectra of CheY and phospho-CheY
The spectrum of unphosphorylated CheY (upper) was obtained just prior to addition of acetyl phosphate, while the phospho-CheY spectrum (lower) was obtained by subtracting out the unphosphorylated component of the CheY/phospho-CheY mixture. This subtraction utilized the 10- and 50-min spectra illustrated in Fig. 6; reaction conditions were as described in the legend to Fig. 6.
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