PDBsum entry 1d4z

Signaling protein

PDB id: 1d4z

Contents

Protein chain

128 a.a. *

Ligands

SO4 ×3

Waters ×141

* Residue conservation analysis

PDB id:

1d4z

Name:

Signaling protein

Title:

Crystal structure of chey-95iv, a hyperactive chey mutant

Structure:

Chemotaxis protein chey. Chain: a. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.90Å R-factor: 0.197 R-free: 0.227

Authors:

M.Schuster,R.Zhao,R.B.Bourret,E.J.Collins

Key ref:

M.Schuster et al. (2000). Correlated switch binding and signaling in bacterial chemotaxis. J Biol Chem, 275, 19752-19758. PubMed id: 10748173 DOI: 10.1074/jbc.M909908199

Date:

06-Oct-99 Release date: 14-Oct-99

Protein chain

P0AE67  (CHEY_ECOLI) -  Chemotaxis protein CheY from Escherichia coli (strain K12)

Seq: 129 a.a.

Struc: 128 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1074/jbc.M909908199

J Biol Chem 275:19752-19758 (2000)

PubMed id: 10748173

Correlated switch binding and signaling in bacterial chemotaxis.

M.Schuster, R.Zhao, R.B.Bourret, E.J.Collins.

ABSTRACT

In Escherichia coli, swimming behavior is mediated by the phosphorylation state of the response regulator CheY. In its active, phosphorylated form, CheY exhibits enhanced binding to a switch component, FliM, at the flagellar motor, which induces a change from counterclockwise to clockwise flagellar rotation. When Ile(95) of CheY is replaced by a valine, increased clockwise rotation correlates with enhanced binding to FliM. A possible explanation for the hyperactivity of this mutant is that residue 95 affects the conformation of nearby residues that potentially interact with FliM. In order to assess this possibility directly, the crystal structure of CheY95IV was determined. We found that CheY95IV is structurally almost indistinguishable from wild-type CheY. Several other mutants with substitutions at position 95 were characterized to establish the structural requirements for switch binding and clockwise signaling at this position and to investigate a general relationship between the two properties. The various rotational phenotypes of these mutants can be explained solely by the amount of phosphorylated CheY bound to the switch, which was inferred from the phosphorylation properties of the mutant CheY proteins and their binding affinities to FliM. Combined genetic, biochemical, and crystallographic results suggest that residue 95 itself is critical in mediating the surface complementarity between CheY and FliM.

Selected figure(s)

Figure 1.
Fig. 1. Binding of FliM peptide to CheY mutants. A and B, titration curves of wild-type CheY ( , circle ), CheY95IA ( , ), CheY95IM ( , ), and CheY95IV ( , ) in the presence and absence of PAM ( filled and open symbols, respectively). The relative decrease in fluorescence intensity upon sequential addition of FliM peptide is shown. The data were fit to a hyperbolic binding function. Note the different scales on the abscissas in A and B. The inset in A shows Eadie-Hofstee plots, which were used to determine dissociation constants (K[D]). The slope of a linear fit to the data yielded K[D]. C, calculated binding affinities. The values shown in the bar graph represent the reciprocal of the respective K[D] values normalized to the K[D] of wild-type CheY in the absence of phosphodonor. White bars denote the absence, and black bars denote the presence of PAM. Binding reactions for CheY13DK and CheY13DK106YW were carried out in the absence of PAM but displayed in black bars because both proteins presumably represent the activated conformation (37, 38).

Figure 2.
Fig. 2. Residue Tyr106 predominantly adopts the outside conformation in CheY95IV instead of the double conformations seen in the wild-type structure (10). Shown here is a stereo view of the final 2F[o] F[c] electron density map (contoured at 1 ) of the Tyr106 side chain for the structure of CheY95IV. The outside conformation of Tyr106 in CheY95IV fits well in this density (shown in black). For comparison, the inside conformation from the wild-type structure is displayed in red. Note that this conformation has a strained C[ ]-C[ ]-C[ ]angle of 135°, which is roughly 20° off the ideal value for this bond angle. Val95 is shown in its two conformations (green and black lines) as inferred from the electron density at this position.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 19752-19758) copyright 2000.