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PDBsum entry 2b7c
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References listed in PDB file
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Key reference
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Title
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Mg2+ and a key lysine modulate exchange activity of eukaryotic translation elongation factor 1b alpha.
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Authors
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Y.R.Pittman,
L.Valente,
M.G.Jeppesen,
G.R.Andersen,
S.Patel,
T.G.Kinzy.
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Ref.
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J Biol Chem, 2006,
281,
19457-19468.
[DOI no: ]
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PubMed id
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Abstract
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To sustain efficient translation, eukaryotic elongation factor B alpha (eEF1B
alpha) functions as the guanine nucleotide exchange factor for eEF1A.
Stopped-flow kinetics using 2'-(or 3')-O-N-methylanthraniloyl (mant)-GDP showed
spontaneous release of nucleotide from eEF1A is extremely slow and accelerated
700-fold by eEF1B alpha. The eEF1B alpha-stimulated reaction was inhibited by
Mg2+ with a K(1/2) of 3.8 mM. Previous structural studies predicted the Lys-205
residue of eEF1B alpha plays an important role in promoting nucleotide exchange
by disrupting the Mg2+ binding site. Co-crystal structures of the lethal K205A
mutant in the catalytic C terminus of eEF1B alpha with eEF1A and eEF1A.GDP
established that the lethality was not due to a structural defect. Instead, the
K205A mutant drastically reduced the nucleotide exchange activity even at very
low concentrations of Mg2+. A K205R eEF1B alpha mutant on the other hand was
functional in vivo and showed nearly wild-type nucleotide dissociation rates but
almost no sensitivity to Mg2+. These results indicate the significant role of
Mg2+ in the nucleotide exchange reaction by eEF1B alpha and establish the
catalytic function of Lys-205 in displacing Mg2+ from its binding site.
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Figure 5.
FIGURE 5. K205A eEF1B  exhibits reduced
exchange activity of eEF1A compared with the WT and K205R forms.
Using stopped-flow kinetics, an eEF1A (1
µM)·mant-GDP (1 µM) complex in binding buffer
containing 5 mM Mg^2+ was rapidly mixed with a solution
containing excess GDP (45 µM) and various concentrations
of eEF1B to reach saturating
conditions: WT (A), K205A (B), or K205R (C). A time course of
fluorescence intensity was monitored for each eEF1B concentration, and data
were fitted to a single or double exponential decay equation to
calculate the dissociation rate constants (  ). A hyperbolic
equation was used to fit the given dissociation rate constants
to calculate the K[d] (micromolar) and k[off](s^-1) values of
K[d] = 4 ± 0.8 and k[off] = 122 ± 8 (A), K[d] =
0.4 ± 0.1 and k[off] = 8 ± 0.3 (B), and K[d] = 2.4
± 0.8 and k[off] = 68 ± 6.6 (C). Residual plots
were prepared to detect experimental error for the fitted data
subsets.
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Figure 6.
FIGURE 6. Mg^2^+ effects on guanine nucleotide exchange.
Using stopped-flow kinetics, an eEF1A (1
µM)·mant-GDP (1 µM) complex in binding buffer
containing the indicated Mg^2+ concentration was rapidly mixed
with a solution containing excess GDP (45 µM) without (A)
or with saturated amounts of eEF1B :10 µM WT (B), 8
µM K205A (C), or 8 µM K205R (D). Binding buffer
without Mg^2+ contained 5 mM EDTA, pH 8.0. A time course of
fluorescence intensity was observed for each Mg^2+
concentration, and data were fitted to a single or double
exponential decay equation to calculate the dissociation rate
constants ( ). A hyperbolic decay
equation was used to fit the given dissociation rate constants
to calculate the apparent K[  ](mM) and k[off](s^-1)
values of K[ ]= 16.5 ± 11.3
and k[off] = 0.2 ± 0.02 (A), K[ ]= 3.8 ± 0.4
and k[off] = 274 ± 6.0 (B), K[ ]= 0.14 ± 0.03
and k[off] = 136 ± 9 (C), and K[ ]= 27 ± 13 and
k[off] = 62.6 ± 3.4 (D). Residual plots were prepared to
detect experimental error for the fitted data subsets.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
19457-19468)
copyright 2006.
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Secondary reference #1
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Title
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Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eef1a:eef1balpha.
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Authors
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G.R.Andersen,
L.Pedersen,
L.Valente,
I.Chatterjee,
T.G.Kinzy,
M.Kjeldgaard,
J.Nyborg.
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Ref.
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Mol Cell, 2000,
6,
1261-1266.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. The Interface between eEF1A Domain 2 and eEF1Bα
Overlaps with aa-tRNA Binding(A) The CCA-aa end of tRNA (gold)
superimposes with two loops of eEF1Bα (gray) when domain 2 (not
shown) of the eEF1A:eEF1Bα complex is superimposed with domain
2 (not shown) of EF-Tu in complex with aa-tRNA.(B) The
flexibility of the loop demonstrated in the NMR structure may
allow aa-tRNA to displace eEF1Bα. The NMR of eEF1Bα is shown
yellow, the X-ray structure in gray, and the CCA-aa end of tRNA
in gold. A space-filling representation of atoms in eEF1A within
10 Å of Phe-163[b] is shown in gray.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #2
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Title
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Crystal structures of nucleotide exchange intermediates in the eef1a-Eef1balpha complex.
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Authors
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G.R.Andersen,
L.Valente,
L.Pedersen,
T.G.Kinzy,
J.Nyborg.
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Ref.
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Nat Struct Biol, 2001,
8,
531-534.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Electron densities around the nucleotides after the
final refinement. a, Sigmaa-weighted 2F[o] - F[c] electron
density map contoured at 1.2  for
the GDP -Mg2+ complex. The 'M' labels a residual electron
density that most likely contains a Mg2+ ion. b, Sigmaa-weighted
2F[o] - F[c] electron density map contoured at 1.2 for
the GDP complex. c, Sigmaa-weighted 2F[o] - F[c] electron
density map contoured at 1.2 for
the GDPNP complex. Water atoms are shown as red spheres. The
electron densities were plotted with the map_cover option in O25
using a radius of 1.5 Å. Labels on residues in eEF1A and eEF1B
 are
red and blue, respectively. All shown atoms were omitted from
map calculations.
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Figure 2.
Figure 2. The exchange mechanism. The shown structures are
EF-Tu -GDP (PDB accession code 1TUI), eEF1A -eEF1B -GDP
-Mg2+ (PDB accession code 1IJF), eEF1A -eEF1B (PDB
accession code 1F60), eEF1A -eEF1B -GDPNP
(PDB accession code 1G7C) and EF-Tu -GDPNP (PDB accession code
1EFT). Regions around the binding site of EF-Tu -GDPNP are
labeled G1 -G5 according to the nomenclature of Sprang28 and
colored identically in the other structures. The G2 region, part
of the switch 1 region, is only shown for the EF-Tu -GDPNP,
because it is distant from the nucleotide in the other
structures. Comparison with EF-Tu -GDP, EF-Tu -GDPNP and EF-Tu
-EF-Ts indicate that the shown peptide in the P-loop of eEF1A is
likely to flip in the exchange reaction. Mg2+ ions are shown as
blue spheres.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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