PDBsum entry: 1djm

Signaling protein

PDB id: 1djm

Contents

Protein chain

129 a.a. *

* Residue conservation analysis

PDB id:

1djm

Name:

Signaling protein

Title:

Solution structure of bef3-activated chey from escherichia coli

Structure:

Chemotaxis protein y. Chain: a. Engineered: yes

Source:

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

NMR struc:

27 models

Authors:

H.S.Cho,S.Y.Lee,D.Yan,X.Pan,J.S.Parkinson,S.Kustu, D.E.Wemmer,J.G.Pelton

Key ref:

H.S.Cho et al. (2000). NMR structure of activated CheY. J Mol Biol, 297, 543-551. PubMed id: 10731410 DOI: 10.1006/jmbi.2000.3595

Date:

03-Dec-99 Release date: 05-Apr-00

Protein chain

P0AE67  (CHEY_ECOLI) -  Chemotaxis protein CheY

Seq: 129 a.a.

Struc: 129 a.a.

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (1 term)
• Biological process: intracellular signal transduction (7 terms)
• Biochemical function: two-component response regulator activity (3 terms)

DOI no: 10.1006/jmbi.2000.3595

J Mol Biol 297:543-551 (2000)

PubMed id: 10731410

NMR structure of activated CheY.

H.S.Cho, S.Y.Lee, D.Yan, X.Pan, J.S.Parkinson, S.Kustu, D.E.Wemmer, J.G.Pelton.

ABSTRACT

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains.

Selected figure(s)

Figure 2.
Figure 2. (a) 15N-1H FHSQC spectrum of BeF[3]-CheY along with (b) superpositions of backbone N, C^a, and C' coordinates for BeF[3]-activated CheY and (c) comparison of BeF[3]-activated and inactive CheY structures shown in stereoview. (a) In the FHSQC, spectrum peaks are labeled with residue numbers. Unassigned backbone resonances are labeled UA. Pairs of side-chain NH[2] resonances are connected by horizontal lines. Signals enclosed in boxes are folded in the 15N dimension. (b) The 27 structures of BeF[3]-activated CheY. Backbone coordinates for residues in the five helices and five-stranded b-sheet were superimposed. (c) Superposition of the 27 structures of BeF[3]-activated CheY (blue), with apo X-ray [Volz and Matsumura 1991] (gold), magnesium-bound X-ray [Bellsolell et al 1994] (red), and mean magnesium-bound NMR [Moy et al 1994] (magenta) structures. Superposition included backbone coordinates for residues in H1, H2, b1, b2, and b3. Considering the mean coordinates obtained from the family of magnesium-bound [Moy et al 1994] and BeF[3]-activated NMR structures, backbone superposition of H1, H2, b1, b2, and b3 yields an r.m.s.d. value of 2.4 Å for the backbone coodinates of residues in H3, b4, H4, b5, and H5. The Figure was produced with the program MOLMOL [Koradi et al 1996]. Uniformly 15N and 15N/13C-labeled samples were prepared by growth in M9 minimal medium supplemented with biotin and either [15N]ammonium chloride or [15N]ammonium chloride and [13C]glucose. The BeF[3]-activated sample conditions were 4 mM CheY, 16 mM BeCl[2], 100 mM NaF, 20 mM MgCl[2], at pH 6.7, and 10 % 2H[2]O. NMR spectra were recorded on AMX 600 and DRX 500 NMR spectrometers at 25 °C. Backbone resonances were assigned with 3D 15N NOESY-FHSQC [Talluri and Wagner 1996], HNCACB [Wittekind and Mueller 1993], CBCA(CO)NH [Grzesiek and Bax 1992a], and HNCA [Grzesiek and Bax 1992b] spectra. Side-chain aliphatic 13C/1H pairs were assigned with 3D 15N TOCSY-HSQC [Driscoll et al 1990], HCCH-TOCSY [Kay et al 1993] and CBCA(CO)NH spectra. In each of the experiments above, purge-type pulsed-field gradients were used to suppress artifacts and the solvent signal [Bax and Pochapsky 1992]. Aromatic assignments were obtained from DQF-COSY [Rance et al 1983] and 13C/1H HMQC spectra [Bax et al 1990]. The assignment process was also aided by making reference to published chemical shifts for CheY [Bruix et al 1993 and Moy et al 1994]. Phi torsion angle restraints were obtained from a 15N HMQC-J spectrum [Kay and Bax 1990]. Stereospecific assignments for Val and Leu methyl groups were obtained by comparison of ct-HSQC spectra of uniformly 13C-labeled and 10 % uniformly 13C-labeled samples [Neri et al 1989 and Szyperski et al 1992]. x1 restraints for the Val, Ile, and Thr residues were obtained from ct-HMQC-J spectra [Grzesiek et al 1993 and Vuister et al 1993a]. NOEs identified in 3D NOESY-FHSQC, 4D 13C/15N HMQC-NOESY-FHSQC and 4D 13C/13C HMQC-NOESY-HMQC (all recorded with a 100 ms mixing time) [Vuister et al 1993b] spectra were classified as strong (2.9 Å upper distance limit), medium (3.3 Å), or weak (5.0 Å). A total of 972 non-trivial NOE restraints (213 intraresidue, 271 sequential, 238 medium-range, and 250 long-range) were used as input to DYANA [Guntert et al 1997], along with 78 phi torsion angle restraints and 17 x1 restraints for the Val, Ile, and Thr residues. Once sets of 20 (of 60) structures reached a backbone r.m.s.d. of 1 Å, 47 hydrogen bonds (94 upper and 94 lower distance restraints (H-O distance restraint 1.8-2.0 Å; N-O 2.7-3.0 Å)), identified on the basis of slow amide proton exchange rates (protection factors greater than 75) and short donor/acceptor distances were included in the calculations. Structures resulting from DYANA calculations with a pseudoatom (van der Waals radius 2.5 Å) corresponding to BeF[3]^ - attached to the side-chain of Asp57 resulted in a backbone r.m.s.d. value of only 0.4 Å when compared to structures without the additional pseudoatom. The 27 of 60 structures (BeF[3]^ - pseudoatom not included) with residual target function values less than 1.0 Å2 (Table 1; target function before energy minimization was 0.3(±0.2) Å2) were subjected to restrained energy minimization using the AMBER94 forcefield [Cornell et al 1995] implemented in the program OPAL [Luginbuhl et al 1996]. Conjugate gradient minimization (1500 steps) included bond, angle, dihedral, improper dihedral, van der Waals, electrostatic, NMR distance, and NMR torsion angle terms. The minimization was performed in a shell of water at least 6 Å thick, with the dielectric constant set to 1, and with no cut-off for non-bonded interactions. PROCHECK analysis [Laskowski et al 1993] of the structures revealed that 99 % of the residues fall within the allowed or generously allowed regions of the Ramachandran map. The 27 energy-minimized structures are used to represent the solution structure of CheY complexed with beryllofluoride and magnesium.

Figure 3.
Figure 3. Ribbon diagrams of CheY in stereo showing movement of side-chains Thr87 and Tyr106 upon activation. Superposition included backbone coordinates for residues in H1, H2, b1, b2, and b3. Relative that depicted in Figure 2, the structures are rotated 90° about a horizontal axis in the page, affording a view (top) of the active site. The loops between b3 and H3 and between H3 and b4 are ill-defined by the NMR data, and should not be used for comparison. (a) CheY taken from the inactive magnesium-bound NMR structure [Moy et al 1994] and (b) representative NMR structure of BeF[3]-activated CheY. Asp57 (blue) is the site of phosphorylation. Highly conserved Tyr106 (green) and Thr87 (red) are also shown. The Thr87 hydroxyl group is represented by a small ball. BeF[3]^ - is modeled as a black ball attached to Asp57. The Figure was created with the program MOLSCRIPT [Kraulis 1991].

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 297, 543-551) copyright 2000.

Figures were selected by the author.