Assessing functional divergence in EF-1alpha and its paralogs in eukaryotes and archaebacteria

Abstract

A number of methods have recently been published that use phylogenetic information extracted from large multiple sequence alignments to detect sites that have changed properties in related protein families. In this study we use such methods to assess functional divergence between eukaryotic EF-1alpha (eEF-1alpha), archaebacterial EF-1alpha (aEF-1alpha) and two eukaryote-specific EF-1alpha paralogs-eukaryotic release factor 3 (eRF3) and Hsp70 subfamily B suppressor 1 (HBS1). Overall, the evolutionary modes of aEF-1alpha, HBS1 and eRF3 appear to significantly differ from that of eEF-1alpha. However, functionally divergent (FD) sites detected between aEF-1alpha and eEF-1alpha only weakly overlap with sites implicated as putative EF-1beta or aminoacyl-tRNA (aa-tRNA) binding residues in EF-1alpha, as expected based on the shared ancestral primary translational functions of these two orthologs. In contrast, FD sites detected between eEF-1alpha and its paralogs significantly overlap with the putative EF-1beta and/or aa-tRNA binding sites in EF-1alpha. In eRF3 and HBS1, these sites appear to be released from functional constraints, indicating that they bind neither eEF-1beta nor aa-tRNA. These results are consistent with experimental observations that eRF3 does not bind to aa-tRNA, but do not support the 'EF-1alpha-like' function recently proposed for HBS1. We re-assess the available genetic data for HBS1 in light of our analyses, and propose that this protein may function in stop codon-independent peptide release.

Figure 1

Phylogenetic analyses. (A) Relationship among aEF-1α, eEF-1α, eRF3^CTD and HBS1^CTD. The open diamond indicates the probable root of the tree. All EF-1α sequence names are omitted. BP values are shown next to major branches. (B) An EF-1β phylogenetic tree. All EF-1β sequence names are omitted except those determined in this study. A BP value for the Eukaryota–Archaebacteria split is shown. Novel sequences determined in this study are indicated by asterisks.

Figure 2

Parametric bootstrap tests for the rate distance across two sub-trees. The histograms indicate the parametric bootstrap null distributions of the absolute-value or non-absolute-value rate distances (abrsum or brsum) between eEF-1α and aEF-1α (abbreviated as Eα vs. Aα) (A and B), between eEF-1β and aEF-1β (Eβ vs. Aβ) (C and D), between eEF-1α and HBS1^CTD (Eα vs. H) (E and F) and between eEF-1α and eRF3^CTD (Eα vs. R) (G and H), respectively. The observed values are indicated by arrows.

Figure 3

Stereo views of the tertiary structure of yeast EF-1α·EF-1β·GDP indicating the location of FD sites. The yeast EF-1α·EF-1β·GDP structure (PDB file 1IJF) is presented with the FD sites detected (top) between eEF-1α and aEF-1α, (middle) between eEF-1α and HBS1^CTD and (bottom) between eEF-1α and eRF3^CTD. The structures of yeast EF-1α and EF-1β are shown by tube representation in gold and silver, respectively, and the GDP molecule is shown in yellow. Positions of RS+ and RS– (type I FD) sites are emphasized in white and red, respectively. Positions with large blue shells are DE, ADE or ΔCP_s (type II FD) sites. Type I FD sites that are also type II FD sites have larger white or red shells.

Figure 4

Plots of NSA values. The plot for the 269 sites analyzed in this study is given as ‘All (269 sites)’. The dotted line represents the median of the NSA values from all analyzed (269) sites. Arrows indicate the medians of each plot. Eα vs. Aα, the FD sites between eEF-1α and aEF-1α; Eα vs. H, the FD sites between eEF-1α and HBS1^CTD; Eα vs. R, the FD sites between eEF-1α and eRF3^CTD.

Figure 5

FD sites detected between the EF-1β orthologs. The RS– and DE sites detected between the EF-1β orthologs are shown in pink and white, respectively. The FD sites detected between the EF-1α orthologs, which are within ∼10 Å centroid-to-centroid distance from the EF-1β FD site (Gly1162 in yeast EF-1β) are presented in green. The residue numbers are based on PDB file 1IJF.

Figure 6

Alignment of the β10 and β11 sheets in eEF-1α and the corresponding region in aEF-1α, HBS1^CTD and eRF3^CTD. The sites included in the analyses are marked by a ‘+’ in the top line. The putative EF-1β and aa-tRNA binding sites are marked by X in the second and third lines, respectively. Yeast EF-1α β10 and β11 sheets (PDB file 1IJF) are shown in boxes in the fourth line. Conserved amino acid residues are shaded.