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PDBsum entry 2vui
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References listed in PDB file
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Key reference
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Title
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The hupr receiver domain crystal structure in its nonphospho and inhibitory phospho states.
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Authors
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K.M.Davies,
E.D.Lowe,
C.Vénien-Bryan,
L.N.Johnson.
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Ref.
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J Mol Biol, 2009,
385,
51-64.
[DOI no: ]
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PubMed id
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Abstract
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Hydrogen uptake protein regulator (HupR) is a member of the nitrogen regulatory
protein C (NtrC) family of response regulators. These proteins activate
transcription by RNA polymerase (RNAP) in response to a change in environment.
This change is detected through the phosphorylation of their receiver domain as
part of a two-component signalling pathway. HupR is an unusual member of this
family as it activates transcription when unphosphorylated, and transcription is
inhibited by phosphorylation. Also, HupR activates transcription through the
more general sigma(70) transcription initiation factor, which does not require
activation by ATPase, in contrast to other NtrC family members that utilise
sigma(54). Hence, its mode of action is expected to be different from those of
the more conventional NtrC family members. We have determined the structures of
the unphosphorylated N-terminal receiver domain of wild-type HupR, the mutant
HupR(D55E)(N) (which cannot be phosphorylated and down-regulated), and HupR in
the presence of the phosphorylation mimic BeF(3)(-). The structures show a
typical response regulator fold organised as a dimer whose interface involves
alpha4-beta5-alpha5 elements. The interactions across the interface are slightly
different between apo and phospho mimics, and these reflect a rearrangement of
key conserved residues around the active site aspartate that have been
implicated in domain activation in other receiver domain proteins. We also show
that the wild-type HupR receiver domain forms a weak dimer in solution, which is
strengthened in the presence of the phosphorylation mimic BeF(3)(-). The results
indicate many features similar to those that have been observed in other
systems, including NtrC (where phosphorylation is activatory), and indicate that
recognition properties, which allow HupR to be active in the absence of
phosphorylation, lie in the transmission of phosphorylation signals through the
linker region to the other domains of the protein.
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Figure 1.
Fig. 1. The structure of HupR[WT]^N. (a) Cartoon
representation of HupR[WT]^N. The site of phosphorylation
(Asp55) is illustrated as green sticks. The position of the
conserved residues involved in activation, Thr83 and Phe103, is
illustrated as yellow and blue sticks, respectively. Secondary
structure elements are labelled as follows: β1, residues
7–12; β2, residues 30–35; β3, residues 48–55; β4,
residues 78–83; β5, residues 101–105; α1, residues
15–28; α2, residues 36–46; α3, residues 63–73; α4,
residues 89–98; α5, residues 109–141. (b) Magnesium binding
at the active site of HupR[WT]^N. Mg^2 + (pink sphere) and H[2]O
(blue sphere). No unexplained density is observed around Asp55;
thus, HupR[WT]^N is unphosphorylated. Blue density corresponds
to the 2F[o] − F[c] map contoured at 1σ, and pink density
corresponds to the F[o] − F[c] map contoured at 5σ, with Mg^2
+ excluded from the phasing. (c) Cartoon representation of a
HupR[WT]^N dimer viewed along the crystallographic 2-fold axis.
The dimer interface occurs between β5 and α5 of neighbouring
monomers. The phosphorylatable aspartate Asp55 is shown as green
sticks, and the conserved aromatic residue Phe103 is shown as
blue sticks. Figures were created with PyMOL™ (DeLano
Scientific LLC).
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Figure 2.
Fig. 2. The structure of HupR[D55E]^N. (a) Overlay of
HupR[WT]^N and HupR[D55E]^N[.] The two structures were aligned
according to residues 6–81. Thr83 and Phe103 are illustrated
as sticks: Thr83-HupR[WT]^N (yellow), Thr83-HupR[D55E]^N
(orange), Phe103-HupR[WT]^N (cyan), and Phe103-HupR[D55E]^N
(light blue). (b) Active site of HupR[WT]^N and HupR[D55E]^N
showing the different conformations of Thr83 and Phe103. In
HupR[WT]^N, Thr83 points away from Asp55, and Phe103 points out
of the monomer. In HupR[D55E]^N, Thr83 forms a hydrogen bond
with Glu55, and Phe103 points into the monomer. (c) Dimer
interface of HupR[WT]^N and HupR[D55E]^N showing the different
interactions that occur across the interface. Hydrophobic
residues: salmon sticks; hydrogen bonds: black dashes.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2009,
385,
51-64)
copyright 2009.
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