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PDBsum entry 1oxx
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Transport protein
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PDB id
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1oxx
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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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Formation of the productive ATP-Mg2+-Bound dimer of glcv, An abc-Atpase from sulfolobus solfataricus.
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Authors
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G.Verdon,
S.V.Albers,
N.Van oosterwijk,
B.W.Dijkstra,
A.J.Driessen,
A.M.Thunnissen.
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Ref.
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J Mol Biol, 2003,
334,
255-267.
[DOI no: ]
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PubMed id
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Abstract
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The ABC-ATPase GlcV from Sulfolobus solfataricus energizes an ABC transporter
mediating glucose uptake. In ABC transporters, two ABC-ATPases are believed to
form a head-to-tail dimer, with both monomers contributing conserved residues to
each of the two productive active sites. In contrast, isolated GlcV, although
active, behaves apparently as a monomer in the presence of ATP-Mg(2+),
AMPPNP-Mg(2+) or ATP alone. To resolve the oligomeric state of the active form
of GlcV, we analysed the effects of changing the putative catalytic base,
residue E166, into glutamine or alanine. Both mutants are, to different extents,
defective in ATP hydrolysis, and gel-filtration experiments revealed their
dimerization in the presence of ATP-Mg(2+). Mutant E166Q forms dimers also in
the presence of ATP alone, without Mg(2+), whereas dimerization of mutant E166A
requires both ATP and Mg(2+). These results confirm earlier reports for other
ABC-ATPases, but for the first time suggest the occurrence of a fast equilibrium
between ATP-bound monomers and ATP-bound dimers. We further mutated two highly
conserved residues of the ABC signature motif, S142 and G144, into alanine. The
G144A mutant is completely inactive and fails to dimerize, indicating an
essential role of this residue in stabilizing the productive dimeric state.
Mutant S142A retained considerable activity, and was able to dimerize, thus
implying that the interaction of the serine with ATP is not essential for
dimerization and catalysis. Furthermore, although the E166A and G144A mutants
each alone are inactive, they produce an active heterodimer, showing that
disruption of one active site can be tolerated. Our data suggest that
ABC-ATPases with partially degenerated catalytic machineries, as they occur in
vivo, can still form productive dimers to drive transport.
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Figure 6.
Figure 6. A, Model of a head-to-tail, ATP-Mg2+-bound dimer
of GlcV. This structure was built by homology modelling at the
SWISS-MODEL server
(http://swissmodel.expasy.org/SWISS-MODEL.html) using as a
template the structure of the ATP-Na^+-bound, LolD dimer.[21.]
The locations of conserved motifs are indicated with colours, as
in Figure 1. The nucleotide and Mg2+ are shown in ball-and-stick
representation. B, Stereo view of one of the two active sites in
the model of the ATP-Mg2+-bound dimer of GlcV. Residue
side-chains, the nucleotide, the Mg2+ and its coordinating water
molecules are shown in ball-and-stick representation. The extra
methyl group in the G144A mutant is shown in cyan, as inferred
from the GlcV-G144A crystal structure, revealing a steric clash
with the oxygen atoms of the g-phosphate group and the
side-chain of S40 (lines indicate the close contacts at 2.3
Å and 1.8 Å, respectively).
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Figure 7.
Figure 7. Proposed mechanism of ATP hydrolysis catalyzed by
GlcV.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2003,
334,
255-267)
copyright 2003.
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