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PDBsum entry 2jb9
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Transcription
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PDB id
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2jb9
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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 X-Ray crystal structures of two constitutively active mutants of the escherichia coli phob receiver domain give insights into activation.
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Authors
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R.Arribas-Bosacoma,
S.K.Kim,
C.Ferrer-Orta,
A.G.Blanco,
P.J.Pereira,
F.X.Gomis-Rüth,
B.L.Wanner,
M.Coll,
M.Solà.
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Ref.
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J Mol Biol, 2007,
366,
626-641.
[DOI no: ]
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PubMed id
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Abstract
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The PhoR/PhoB two-component system is a key regulatory protein network enabling
Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by
turning on Pho regulon genes for more efficient Pi uptake and use of alternative
phosphorus sources. Under environmental Pi depletion, the response regulator
(RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process
that requires Mg(2+) bound at the active site. Phosphorylation of the RD
relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region
that binds to Pho regulon promoters to activate transcription. The molecular
details of the activation are proposed to involve dimerization of the RD and a
conformational change in the RD detected by the ED. The structure of the PhoB RD
shows a symmetrical interaction involving alpha1, loop beta5alpha5 and N
terminus of alpha5 elements, also seen in the complex of PhoB RD with Mg(2+), in
which helix alpha4 highly increases its flexibility. PhoB RD in complex with
Mg(2+) and BeF(3) (an emulator of the phosphate moiety) undergoes a dramatic
conformational change on helix alpha4 and shows another interaction involving
alpha4, beta5 and alpha5 segments. We have selected a series of constitutively
active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon
promoters in the absence phosphorylation and, as they cannot be inactivated,
should therefore mimic the active RD state of PhoB and its functional
oligomerisation. We have analysed the PhoB(CA) RD crystal structures of two such
mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants
reproduce the homodimeric arrangement through the symmetric interface
encountered in the unbound and magnesium-bound wild-type PhoB RD structures.
Besides, the mutant RD structures show a modified active site organization as
well as changes at helix alpha4 that correlate with repositioning of surrounding
residues, like the active-site events indicator Trp54, putatively redifining the
interaction with the ED in the full-length protein.
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Figure 1.
Figure 1. (a) The structure of PhoB RD. Richardson diagram of
PhoB RD. Helices are shown as blue ribbons (α1 to α5) and
β-strands as yellow arrows (β1 to β5). The N and C-terminal
ends are indicated (N and C, respectively). The green arrow
points to the active site cavity. Key residue side-chains
mentioned throughout the text are highlighted. (b) Dimers
interacting through the α1-Lβ5α5 interface. Cα plot showing
the superimposition of WT-RD (grey) and DAYC-Na (khaki) dimers.
Secondary structure elements belonging to the interface are
indicated; the distance between vicinal Lβ4α4 loops within a
dimer is shown. The green arrows point to the active site
cavities of each protomer within a dimer. The C and N-terminal
ends of the RDs are shown (N and C, respectively). In each case,
molecules A are on the left and molecules B on the right of the
dimer. (c) Packing of WT (grey) and DAYC (khaki) helix α4
against the protein core. Mutated residues or side-chains that
change the conformation are indicated twice, with colour coding
according the molecule they belong to. α-helices and β-strands
numbering is indicated. (d) Each constituting monomer within the
WT RD dimer (superimposed with its solid Connolly surface) has
been rotated vertically 90° from the position occupied in
(b) to grant insight into the interacting surface. The residues
of each molecule participating in direct contacts are shown and
labelled. The colour coding reflects conservation of each
position, ranging from 0% (light yellow) to 100% (intense
red). The approximate location of the active site cleft is
indicated by a green arrowhead. (e) DADE (green) and
BeF-RD[3]^36 (coral) active sites are superimposed (strands β1,
β3 and β4 where used for an optimal superposition of residues
in the active site). Mutated residues or side-chains that change
the conformation are indicated twice. The small spheres
represent water (W) molecules, which are numbered according to
the PDB. The M sphere represents the coordinated metal; the Be
sphere the Beryllium and F1, F2 and F3 the fluorine atoms in the
BeF[3]¯ molecule, an analogue of the activating phosphate
moiety. Electrostatic and hydrogen bond interactions are
represented by broken lines and, together with water oxygen
atoms, are coloured according to the PDB they belong to. W3 and
the interaction between the cation and Met55 carbonyl are not
shown for clarity. Secondary structure elements are indicated.
(f) DAYC (khaki) and BeF[3]-RD^36 (coral) are superimposed
around their active sites. The criteria of representation are
the same as for (e).
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The above figure is
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
366,
626-641)
copyright 2007.
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Secondary reference #1
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Title
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Three-Dimensional crystal structure of the transcription factor phob receiver domain.
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Authors
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M.Solá,
F.X.Gomis-Rüth,
L.Serrano,
A.González,
M.Coll.
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Ref.
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J Mol Biol, 1999,
285,
675-687.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. (a) Ribbon diagram (MOLMOL; [Koradi et al 1996])
showing the structure of the PhoB receiver domain. The two
molecules in the asymmetric unit are depicted with the local
dyad vertical in this view. Note that the loop α4β4, at the
top of the Figure, does not participate in inter-protein
contacts although being located in the same face of the
interacting surface. (b) Protein-protein interface
representation (BOBSCRIPT; [Esnouf 1997]) viewed along the dyad
axis. Residues and secondary structure elements at the interface
are shown. Lys105 side-chain is also represented in order to
show the connection between the active site and the presumed
dimerization surface.
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Figure 4.
Figure 4. Stereo view of the active site of (a) molecule A
and (b) B (drawn with MOLMOL; [Koradi et al 1996]), showing
important residues. Hydrogen bonds are depicted with broken
lines. Water molecules as blue spheres. Note, in molecule A, the
connection of loop β4α4 and helix α4 with the active site,
through Lys105 main-chain and water-bridge Glu88-W5-Asp53.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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