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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Crystal structure of activated chey. Comparison with other activated receiver domains.
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Authors
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S.Y.Lee,
H.S.Cho,
J.G.Pelton,
D.Yan,
E.A.Berry,
D.E.Wemmer.
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Ref.
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J Biol Chem, 2001,
276,
16425-16431.
[DOI no: ]
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PubMed id
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Abstract
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The crystal structure of BeF(3)(-)-activated CheY, with manganese in the
magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to
Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt
bridge with Thr(87) and Lys(109), respectively. The six coordination sites for
manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of
Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules.
All of the active site interactions seen for BeF(3)(-)-CheY are also observed in
P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by
mimicking both the tetrahedral geometry and electrostatic potential of a
phosphoryl group. The aromatic ring of Tyr(106) is found buried within a
hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side
chain is stabilized in this conformation by a hydrogen bond between the hydroxyl
group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to
stabilize the active conformation of the beta4/H4 loop. Comparison of the
backbone coordinates for the active and inactive states of CheY reveals that
only modest changes occur upon activation, except in the loops, with the largest
changes occurring in the beta4/H4 loop. This region is known to be
conformationally flexible in inactive CheY and is part of the surface used by
activated CheY for binding its target, FliM. The pattern of activation-induced
backbone coordinate changes is similar to that seen in FixJ(r). A common feature
in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY
is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent,
beryllofluoride. This suggests that, in addition to the concerted movements of
Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt
bridge is directly involved in the activation of receiver domains generally.
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Figure 1.
Fig. 1. Ribbon diagram of the two BeF[  -
]-activated CheY molecules in the asymmetric unit. The active
sites are directed toward the reader. Side chains are shown for
BeF[  -
]-Asp57, Thr87, and Tyr106.
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Figure 3.
Fig. 3. Stereo view of the active site of BeF[ -
]-CheY. Carbon, nitrogen, oxygen beryllofluoride, and manganese
atoms are colored gray, dark blue, red, yellow, and green,
respectively. a, omit map contoured at 3.0  covering
Asp12, Asp13, BeF[ -
]-Asp57, Thr87, Lys109, and two water molecules. This map was
calculated with the occupancies for these residues set to zero.
For clarity, the density for manganese is not shown. b,
ball-and-stick diagram of the BeF[ -
]-activated CheY active site. Dashed lines and numbers denote
active site interactions defined in Table II. c, stereo view of
active site residues for BeF[ -
]-CheY(Mn2+) (blue), phosphorylated FixJr(no metal) (lime), and
phosphorylated Spo0A^r(Ca^2+) (copper). Mn2+ and Ca^2+ are shown
as red and green balls, respectively. Residue numbers are based
on E. coli CheY. For clarity, phosphono-CheY was not included.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2001,
276,
16425-16431)
copyright 2001.
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Secondary reference #1
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Title
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Nmr structure of activated chey.
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Authors
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H.S.Cho,
S.Y.Lee,
D.Yan,
X.Pan,
J.S.Parkinson,
S.Kustu,
D.E.Wemmer,
J.G.Pelton.
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Ref.
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J Mol Biol, 2000,
297,
543-551.
[DOI no: ]
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PubMed id
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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.
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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].
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #2
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Title
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Phosphorylated aspartate in the structure of a response regulator protein.
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Authors
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R.J.Lewis,
J.A.Brannigan,
K.Muchová,
I.Barák,
A.J.Wilkinson.
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Ref.
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J Mol Biol, 1999,
294,
9.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. The active site of N-Spo0A
~
phosphate. (a) Stereo view of the initial electron density map displayed on
selected atoms from the final refined model in the vicinity of the aspartyl phosphate (left). The map is calculated using
coefficients 2Fobs
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Fcalc using 4-fold non-crystallographic symmetry-averaged density modified phases from the initial
molecular replacement solution. In these unbiased maps, electron density defining the phosphoryl group, the calcium
ion (green) and neighbouring water molecules is clear. (b) Stereo view of an Fobs
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Fcalc electron density omit map con-
toured at 3.5s displayed around Asp55, the phosphoryl group and the calcium ion in the refined co-ordinate set. This
map was calculated with the occupancies of the side-chain atoms of residue 55, the calcium ion and the nearby water
molecules set to zero. Atoms are coloured according to type; C (cyan), O (red), N (blue), P (yellow), Ca (grey) and water
(green). (c) Stereo view orthogonal to (b). Hydrogen bonds are shown as broken lines, AP denotes the phosphorylated
Asp. The calcium ion is seven co-ordinate, its ligands being three water molecules, carboxylate oxygen atoms of Asp10
and Asp55, a phosphoryl oxygen atom and the main-chain carbonyl oxygen atoms of Ile57. The phosphoryl oxygen
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Figure 3.
Figure 3. Concerted movements of active site Thr and
aromatic side-chains on phosphorylation. (a) Compari-
son of the active sites of N-Spo0A
~
phosphate (cyan
with phosphorus in yellow), Spo0F (green) and CheY
(pink). Oxygen atoms are coloured in red and the
hydroxyl oxygen atoms of the threonine residues have
been slightly enlarged. The opposing directions of the
threonine side-chain in the presence and absence of the
phosphoryl group are apparent. (b) A comparison of
Asp55, Thr84 and Phe103 of Ca
2+
-bound N-Spo0A
~
phosphate (cyan) with the corresponding Asp57,
Thr87 and Tyr106 of Mg
2+
-coordinated CheY (pink),
emphasising the concerted movements upon
phosphorylation. The structures were superimposed on
the main-chain atoms of 112 residues with an rmsdelta of
1.5 Å .
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #3
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Title
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Conformational changes induced by phosphorylation of the fixj receiver domain.
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Authors
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C.Birck,
L.Mourey,
P.Gouet,
B.Fabry,
J.Schumacher,
P.Rousseau,
D.Kahn,
J.P.Samama.
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Ref.
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Structure, 1999,
7,
1505-1515.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. View of the phosphorylated FixJN dimer. Each
protomer is represented by ribbons and the color varies from
dark blue (N terminus) to green (C terminus). The secondary
structure elements are labeled and the phosphoaspartate groups
are shown as spheres. The van der Waals surface of the dimer
generated using SURFNET [58] is shown as a transparent solid.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #4
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Title
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The 1.9 a resolution crystal structure of phosphono-Chey, An analogue of the active form of the response regulator, Chey.
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Authors
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C.J.Halkides,
M.M.Mcevoy,
E.Casper,
P.Matsumura,
K.Volz,
F.W.Dahlquist.
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Ref.
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Biochemistry, 2000,
39,
5280-5286.
[DOI no: ]
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PubMed id
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