Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering - PubMed (original) (raw)

. 1993 Jun 25;268(18):13089-96.

Affiliations

PMID: 8514750
PMCID: PMC2892986

Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering

R B Bourret et al. J Biol Chem. 1993.

Abstract

The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phosphorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational "switch" in the activation site, is required for activation of CheY by either phosphorylation or mutation. The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch.

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Figures

Fig. 1. Structure of the CheY activation site and location of selected residues

A, the activation site includes Asp12, Asp13 (site of activating mutations), Asp57 (site of phosphorylation), and Lys109 (putative conformational switch). 4F-Phe reporter group locations for the carboxylate cluster (Phe14) and Lys109 (Phe111) in 19F NMR experiments are also indicated. B, the face of CheY that binds to the flagellar switch has been proposed to include residues Leu24, Glu27, Ala90, Ala99, Ser104, Val108, Pro110, Phe111, Thr112, Ala113, Thr115, and Glu117 (unlabeled side chains) (23, 32). In addition, Ala88 (stippled) and Lys109, which are contiguous with this surface, are implicated by the present study.

Fig. 2. Effect of mutations that perturb the Asp57-Lys109 interaction on the 19F NMR spectra of 4F-Phe-labeled CheY

Each spectral pair consists of the spectrum from wild-type CheY (fine line) overlaid on the spectrum from the indicated mutant (bold line). 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 _μ_m 5-fluorotryptophan as internal frequency standard, 25 °C. Assignments are from the preceding paper (Ref. 5, Fig. 3).

Fig. 3. Effect of control activation site mutations on the 19F NMR spectra of 4F-Phe-labeled CheY

Each spectral pair consists of the spectrum from wild-type CheY (fine line) overlaid on the spectrum from the indicated mutant (bold line). Sample parameters were as described in the legend to Fig. 2.

Fig. 4. Use of Gd(III) EDTA to assign the 4F-Phe14 19F NMR resonance from 4F-Phe-labeled CheYD13K

Spectra taken in the absence (fine line) or presence (bold line) of the aqueous paramagnetic probe Gd(III)·EDTA are displayed. 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. Effect of activating mutations on the 19F NMR spectra of 4F-Phe-labeled CheY

Each spectral pair consists of the spectrum from wild-type CheY (fine line) overlaid with the spectrum from the indicated mutant (bold line). Assignment of the 4F-Phel4 resonance, determined with Gd(III)·EDTA, is indicated by the asterisk. Sample parameters were as described in the legend to Fig. 2.

Fig. 6. Effect of mutations perturbing the Phe cluster on the 19F NMR spectra of 4F-Phe-labeled CheY

Each spectral pair consists of the spectrum from wild-type CheY (fine line) overlaid with the spectrum from the indicated mutant (bold line). Sample parameters were as described in the legend to Fig. 2.

Fig. 7. Two-step working model of CheY activation

The indicated schematic model accounts for observations described in this and the preceding paper (5). See text for detailed discussion. A, activation by phosphorylation. Shown are the Asp57 and Lys109 side chain charges in the activation site of wild-type CheY. Upon phosphorylation of Asp57, the Lys109 side chain retains its original conformation, perhaps forming a salt bridge with the phosphoryl group. When phospho-CheY docks to the flagellar switch, a Mg(II) ion binds to the phosphoryl group and breaks any remaining electrostatic interaction between Lys109 and the acylphosphate. The Mg(II) and repositioned Lys109 interact with the switch to cause CW flagellar rotation. B, activation by mutation. A positively charged Lys or Arg side chain at position 13 mimics the position of the surface Mg(II) in phosphorylated wild-type CheY. This positive charge also disrupts the Asp57-Lys109 electrostatic interaction, together yielding partial activation.

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Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering - PubMed (original) (raw)