PDBsum entry 2b7c

Translation

PDB id: 2b7c

Contents

Protein chains

437 a.a. *

90 a.a. *

Waters ×536

* Residue conservation analysis

PDB id:

2b7c

Name:

Translation

Title:

Yeast guanine nucleotide exchange factor eef1balpha k205a mutant in complex with eef1a

Structure:

Elongation factor 1-alpha. Chain: a. Synonym: ef-1-alpha, translation elongation factor 1a, eukaryotic elongation factor 1a, eef1a, elongation factor 1a. Elongation factor-1 beta. Chain: b. Fragment: c-terminal domain. Synonym: ef-1-beta, translation elongation factor 1b alpha, eukaryotic elongation factor 1balpha, eef1balpha, elongation factor

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: tef5. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.210 R-free: 0.236

Authors:

Y.R.Pittman,L.Valente,M.G.Jeppesen,G.R.Andersen,S.Patel,T.G.Kinzy

Key ref:

Y.R.Pittman et al. (2006). Mg2+ and a key lysine modulate exchange activity of eukaryotic translation elongation factor 1B alpha. J Biol Chem, 281, 19457-19468. PubMed id: 16675455 DOI: 10.1074/jbc.M601076200

Date:

04-Oct-05 Release date: 02-May-06

Protein chain

P02994  (EF1A_YEAST) -  Elongation factor 1-alpha from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 458 a.a.

Struc: 437 a.a.

Protein chain

P32471  (EF1B_YEAST) -  Elongation factor 1-beta from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 206 a.a.

Struc: 90 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M601076200

J Biol Chem 281:19457-19468 (2006)

PubMed id: 16675455

Mg2+ and a key lysine modulate exchange activity of eukaryotic translation elongation factor 1B alpha.

Y.R.Pittman, L.Valente, M.G.Jeppesen, G.R.Andersen, S.Patel, T.G.Kinzy.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 19457-19468) copyright 2006.

Figures were selected by the author.