Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca2+-calmodulin

Abstract

Background: Eukaryotic translation elongation factor eEF1A directs the correct aminoacyl-tRNA to ribosomal A-site. In addition, eEF1A is involved in carcinogenesis and apoptosis and can interact with large number of non-translational ligands. There are two isoforms of eEF1A, which are 98% similar. Despite the strong similarity, the isoforms differ in some properties. Importantly, the appearance of eEF1A2 in tissues in which the variant is not normally expressed can be coupled to cancer development.We reasoned that the background for the functional difference of eEF1A1 and eEF1A2 might lie in changes of dynamics of the isoforms.

Results: It has been determined by multiple MD simulation that eEF1A1 shows increased reciprocal flexibility of structural domains I and II and less average distance between the domains, while increased non-correlated diffusive atom motions within protein domains characterize eEF1A2. The divergence in the dynamic properties of eEF1A1 and eEF1A2 is caused by interactions of amino acid residues that differ between the two variants with neighboring residues and water environment. The main correlated motion of both protein isoforms is the change in proximity of domains I and II which can lead to disappearance of the gap between the domains and transition of the protein into a "closed" conformation. Such a transition is reversible and the protein can adopt an "open" conformation again. This finding is in line with our earlier experimental observation that the transition between "open" and "closed" conformations of eEF1A could be essential for binding of tRNA and/or other biological ligands. The putative calmodulin-binding region Asn311-Gly327 is less flexible in eEF1A1 implying its increased affinity for calmodulin. The ability of eEF1A1 rather than eEF1A2 to interact with Ca2+/calmodulin is shown experimentally in an ELISA-based test.

Conclusion: We have found that reversible transitions between "open" and "close" conformations of eEF1A provide a molecular background for the earlier observation that the eEF1A molecule is able to change the shape upon interaction with tRNA. The ability of eEF1A1 rather than eEF1A2 to interact with calmodulin is predicted by MD analysis and showed experimentally. The differential ability of the eEF1A isoforms to interact with signaling molecules discovered in this study could be associated with cancer-related properties of eEF1A2.

Figure 1

Alignment of the sequences of elongation factors 1A. eEF1A1 – human eEF1A1 [Swiss-Prot: P68104], eEF1A2 – human eEF1A2 [Swiss-Prot: Q05639], Yeast – yeast eEF1A [Swiss-Prot: P02994], S. Solfataricus – aEF1A of Sulfolobus solfataricus [Swiss-Prot: P35021], T. Aquaticus – EF-Tu of Termus Aquaticus [Swiss-Prot: Q01698]. Replacements and deletion in eEF1A1 and eEF1A2 are marked in red.

Figure 2

Ribbon representation of the eEF1A molecule. a – domains I, II and III; 1 and 2 – amino acid residues Arg69-Leu77 and His295-Gly305, which are on the surface of the gap between the domains I and II, 3 and 4 – motifs Asn311-Gly327 and Gly422-Val437, suggested to be the calmodulin binding site in eEF1A1. b – amino acid residues with a positive difference between the rmsf of eEF1A2 and eEF1A1 of more than 0.02 nm (Met1-Lys5, Asp35-Glu48, Gly50-Thr58, Asp61, Lys62, Lys64-Glu68, Gly70-Asp74, Ala125-Ser128, Val216-Gly221, Ala223, Cys/Thr234, Gly258, Ile259, Pro282, His296-Ala298, Ser316, M3l318-Arg322, Gln352, His364, Thr365, Tyr418-Pro420, ribbon colored in red) and with negative differences of less than -0.02 nm (Asn130, Ser157, Lys313, Ser329, Ser383, Gly384, Glu388, Asp398, ribbon colored in blue); side chains of residues Asp197 and Mly55 which stabilize the motif Asp35-Asp74 in eEF1A1 are shown.

Figure 3

Distance between centers of domains I and II (a-d) and minimal distance between regions Arg69-Leu77 and His295-Gly305 (e, f). a, e – eEF1A1: red – trajectory 1, green – trajectory 2, blue – trajectory 3, cyan – trajectory 4, magenta – trajectory 5, yellow – trajectory 6. b, f – eEF1A2: red – trajectory 7, green – trajectory 8, blue – trajectory 9, cyan – trajectory 10, magenta – trajectory 11, yellow – trajectory 12, orange – trajectory 13. c – the mean distances between centers of the domains I and II for eEF1A1 (black) and eEF1A2 (red); averaging has been done for all respective trajectories. d – the mean distances between the centers of domains I and II after the exemption of the trajectories characterized by the existence of "closed" conformation for more than 500 ps (1, 6 for eEF1A1 and 7, 8, 13 for eEF1A2).

Figure 4

Main correlated motions of two isoforms of human translation elongation factor 1A. a-i – eEF1A1, j-r – eEF1A2. a-c – 2500–10466 ps of trajectory 2; d-f – 5440–10514 ps of trajectory 4; g-i – 3960–9920 of trajectory 5; j-l – 1070–10000 ps of trajectory 9; m-o – 3780–10417 ps of trajectory 11; p-r – 1130–10197 ps of trajectory 12. a, d, g, j, m, p – first eigenvector; b, e, h, k, n, q – second eigenvector; c, f, i, l, o, r – third eigenvector. The protein regions of the maximal C_α-atom displacements (> 0.05 nm) are colored in black. The motions are shown on the average protein conformations in the respective trajectory ranges.

Figure 5

Root-mean-square fluctuations of C-alpha atoms of the two eEF1A isoforms. Data are averaged for the trajectories 1–6 of eEF1A1 and 7–9, 11–13 of eEF1A2. A – rmsf of eEF1A1 (black) and eEF1A2 (gray). B – difference between rmsf of eEF1A2 and eEF1A1. I – domain I, II – domain II, III – domain III.

Figure 6

Conservation of exposed residues in putative calmodulin binding domain of the eEF1A homologues.

Figure 7

C_α-atoms rmsd of Asn311-Gly327 after fitting of domains II to the initial domain conformation. a – eEF1A1: red – trajectory 1, green – trajectory 2, blue – trajectory 3, cyan – trajectory 4, magenta – trajectory 5, yellow – trajectory 6. b – eEF1A2: red – trajectory 7, green – trajectory 8, blue – trajectory 9, cyan – trajectory 10, magenta – trajectory 11, yellow – trajectory 12, orange – trajectory 13. Black – average curves.

Figure 8

Comparison of Ca²⁺-calmodulin binding to eEF1A isoforms by enzyme-linked immunosorbent assay-based binding assay. 1 – eEF1A1, 2 – eEF1A2. Microtiter 96-well plates (Dynatech microtiter) were coated with purified eEF1A1 or eEF1A2, and monoclonal anti-eEF1A antibody was added with or without increasing amounts of Ca²⁺-calmodulin.