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PDBsum entry 1d4z
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Signaling protein
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PDB id
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1d4z
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Contents |
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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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Correlated switch binding and signaling in bacterial chemotaxis.
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Authors
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M.Schuster,
R.Zhao,
R.B.Bourret,
E.J.Collins.
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Ref.
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J Biol Chem, 2000,
275,
19752-19758.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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).
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
19752-19758)
copyright 2000.
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