Structure of the constitutively active double mutant CheY D13K Y106W alone and in complex with a FliM peptide

Collin M. Dyer, Michael L. Quillin, Andres Campos, Justine Lu, Megan McEvoy, Andrew Hausrath, Edwin M. Westbrook, Philip Matsumura, Brian W. Matthews, Frederick W. Dahlquist

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria. The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro. The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown. We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM 16) derived from the flagellar motor switch, FliM, to 1.5 Å resolution with an R-factor of 19.6%. The asymmetric unit contains four CheY** molecules, two with FliM 16 bound, and two without. The two CheY** molecules in the asymmetric unit that are bound to FliM 16 adopt a conformation similar to BeF 3 --activated wild-type CheY, and also bind FliM 16 in a nearly identical manner. The CheY** molecules that do not bind FliM 16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations. Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM.

Original languageEnglish (US)
Pages (from-to)1325-1335
Number of pages11
JournalJournal of Molecular Biology
Volume342
Issue number4
DOIs
StatePublished - Sep 24 2004
Externally publishedYes

Fingerprint

R388
Phosphorylation
Selenomethionine
Peptides
Proteins
Ligands
Bacteria
Phenotype
In Vitro Techniques

Keywords

  • activating mutation
  • chemotaxis
  • CheY
  • FliM
  • protein-protein interactions

ASJC Scopus subject areas

  • Virology

Cite this

Structure of the constitutively active double mutant CheY D13K Y106W alone and in complex with a FliM peptide. / Dyer, Collin M.; Quillin, Michael L.; Campos, Andres; Lu, Justine; McEvoy, Megan; Hausrath, Andrew; Westbrook, Edwin M.; Matsumura, Philip; Matthews, Brian W.; Dahlquist, Frederick W.

In: Journal of Molecular Biology, Vol. 342, No. 4, 24.09.2004, p. 1325-1335.

Research output: Contribution to journalArticle

Dyer, CM, Quillin, ML, Campos, A, Lu, J, McEvoy, M, Hausrath, A, Westbrook, EM, Matsumura, P, Matthews, BW & Dahlquist, FW 2004, 'Structure of the constitutively active double mutant CheY D13K Y106W alone and in complex with a FliM peptide', Journal of Molecular Biology, vol. 342, no. 4, pp. 1325-1335. https://doi.org/10.1016/j.jmb.2004.07.084
Dyer, Collin M. ; Quillin, Michael L. ; Campos, Andres ; Lu, Justine ; McEvoy, Megan ; Hausrath, Andrew ; Westbrook, Edwin M. ; Matsumura, Philip ; Matthews, Brian W. ; Dahlquist, Frederick W. / Structure of the constitutively active double mutant CheY D13K Y106W alone and in complex with a FliM peptide. In: Journal of Molecular Biology. 2004 ; Vol. 342, No. 4. pp. 1325-1335.
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AU - Lu, Justine

AU - McEvoy, Megan

AU - Hausrath, Andrew

AU - Westbrook, Edwin M.

AU - Matsumura, Philip

AU - Matthews, Brian W.

AU - Dahlquist, Frederick W.

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AB - CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria. The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro. The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown. We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM 16) derived from the flagellar motor switch, FliM, to 1.5 Å resolution with an R-factor of 19.6%. The asymmetric unit contains four CheY** molecules, two with FliM 16 bound, and two without. The two CheY** molecules in the asymmetric unit that are bound to FliM 16 adopt a conformation similar to BeF 3 --activated wild-type CheY, and also bind FliM 16 in a nearly identical manner. The CheY** molecules that do not bind FliM 16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations. Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM.

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