Background Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome. Methodology/Principal Findings To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Bα and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins. Conclusions The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis.
References
[1]
Lund A, Knudsen SM, Vissing H, Clark B, Tommerup N (1996) Assignment of human elongation factor 1alpha genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3. Genomics 36: 359–361.
[2]
Lee S, Francoeur AM, Liu S, Wang E (1992) Tissue-specific expression in mammalian brain, heart, and muscle of S1, a member of the elongation factor-1 alpha gene family. J Biol Chem 267: 24064–24068.
[3]
Chambers DM, Peters J, Abbott CM (1998) The lethal mutation of the mouse wasted (wst) is a deletion that abolishes expression of a tissue-specific isoform of translation elongation factor 1alpha, encoded by the Eef1a2 gene. Proc Natl Acad Sci U S A 95: 4463–4468.
[4]
Newbery HJ, Loh DH, O'Donoghue JE, Tomlinson VA, Chau YY, et al. (2007) Translation elongation factor eEF1A2 is essential for post-weaning survival in mice. J Biol Chem 282: 28951–28959.
[5]
Kahns S, Lund A, Kristensen P, Knudsen CR, Clark BF, et al. (1998) The elongation factor 1 A-2 isoform from rabbit: cloning of the cDNA and characterization of the protein. Nucleic Acids Res 26: 1884–1890.
[6]
Mansilla F, Friis I, Jadidi M, Nielsen KM, Clark BF, et al. (2002) Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system. Biochem J 365: 669–676.
[7]
Le Sourd F, Boulben S, Le Bouffant R, Cormier P, Morales J, et al. (2006) eEF1B: At the dawn of the 21st century. Biochimica Et Biophysica Acta-Gene Structure and Expression 1759: 13–31.
[8]
Ejiri S (2002) Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated nuclear localization. Biosci Biotechnol Biochem 66: 1–21.
[9]
Murray JW, Edmonds BT, Liu G, Condeelis J (1996) Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends. J Cell Biol 135: 1309–1321.
[10]
Duttaroy A, Bourbeau D, Wang XL, Wang E (1998) Apoptosis rate can be accelerated or decelerated by overexpression or reduction of the level of elongation factor-1 alpha. Exp Cell Res 238: 168–176.
[11]
Khacho M, Mekhail K, Pilon-Larose K, Pause A, Cote J, et al. (2008) eEF1A is a novel component of the mammalian nuclear protein export machinery. Mol Biol Cell 19: 5296–5308.
[12]
Chuang SM, Chen L, Lambertson D, Anand M, Kinzy TG, et al. (2005) Proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1A. Mol Cell Biol 25: 403–413.
[13]
Shamovsky I, Ivannikov M, Kandel ES, Gershon D, Nudler E (2006) RNA-mediated response to heat shock in mammalian cells. Nature 440: 556–560.
[14]
Tatsuka M, Mitsui H, Wada M, Nagata A, Nojima H, et al. (1992) Elongation factor-1 alpha gene determines susceptibility to transformation. Nature 359: 333–336.
[15]
Jeganathan S, Morrow A, Amiri A, Lee JM (2008) Eukaryotic elongation factor 1A2 cooperates with phosphatidylinositol-4 kinase III beta to stimulate production of filopodia through increased phosphatidylinositol-4,5 bisphosphate generation. Mol Cell Biol 28: 4549–4561.
[16]
Schlaeger C, Longerich T, Schiller C, Bewerunge P, Mehrabi A, et al. (2008) Etiology-dependent molecular mechanisms in human hepatocarcinogenesis. Hepatology 47: 511–520.
[17]
Anand N, Murthy S, Amann G, Wernick M, Porter LA, et al. (2002) Protein elongation factor EEF1A2 is a putative oncogene in ovarian cancer. Nat Genet 31: 301–305.
[18]
Tomlinson VA, Newbery HJ, Wray NR, Jackson J, Larionov A, et al. (2005) Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours. BMC Cancer 5: 113.
[19]
Cao H, Zhu Q, Huang J, Li B, Zhang S, et al. (2009) Regulation and functional role of eEF1A2 in pancreatic carcinoma. Biochem Biophys Res Commun 380: 11–16.
[20]
Li R, Wang H, Bekele BN, Yin Z, Caraway NP, et al. (2006) Identification of putative oncogenes in lung adenocarcinoma by a comprehensive functional genomic approach. Oncogene 25: 2628–2635.
[21]
Tomlinson VA, Newbery HJ, Bergmann JH, Boyd J, Scott D, et al. (2007) Expression of eEF1A2 is associated with clear cell histology in ovarian carcinomas: overexpression of the gene is not dependent on modifications at the EEF1A2 locus. Br J Cancer 96: 1613–1620.
[22]
Newbery HJ, Gillingwater TH, Dharmasaroja P, Peters J, Wharton SB, et al. (2005) Progressive loss of motor neuron function in wasted mice: effects of a spontaneous null mutation in the gene for the eEF1 A2 translation factor. J Neuropathol Exp Neurol 64: 295–303.
[23]
Lutsep HL, Rodriguez M (1989) Ultrastructural, morphometric, and immunocytochemical study of anterior horn cells in mice with “wasted” mutation. J Neuropathol Exp Neurol 48: 519–533.
[24]
Nagashima K, Kasai M, Nagata S, Kaziro Y (1986) Structure of the two genes coding for polypeptide chain elongation factor 1 alpha (EF-1 alpha) from Saccharomyces cerevisiae. Gene 45: 265–273.
[25]
Mita K, Morimyo M, Ito K, Sugaya K, Ebihara K, et al. (1997) Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors. Gene 187: 259–266.
[26]
Rush J, Moritz A, Lee KA, Guo A, Goss VL, et al. (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23: 94–101.
[27]
Rikova K, Guo A, Zeng Q, Possemato A, Yu J, et al. (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131: 1190–1203.
[28]
Molina H, Horn DM, Tang N, Mathivanan S, Pandey A (2007) Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 104: 2199–2204.
[29]
Guo A, Villen J, Kornhauser J, Lee KA, Stokes MP, et al. (2008) Signaling networks assembled by oncogenic EGFR and c-Met. Proc Natl Acad Sci U S A 105: 692–697.
[30]
Zhu H, Lam DC, Han KC, Tin VP, Suen WS, et al. (2007) High resolution analysis of genomic aberrations by metaphase and array comparative genomic hybridization identifies candidate tumour genes in lung cancer cell lines. Cancer Lett 245: 303–314.
[31]
Panasyuk G, Nemazanyy I, Filonenko V, Negrutskii B, El'skaya AV (2008) A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules. Int J Biochem Cell Biol 40: 63–71.
[32]
Lamberti A, Longo O, Marra M, Tagliaferri P, Bismuto E, et al. (2007) C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A. Cell Death Differ 14: 952–962.
[33]
Eckhardt K, Troger J, Reissmann J, Katschinski DM, Wagner KF, et al. (2007) Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1. Cellular Physiology and Biochemistry 20: 227–240.
[34]
Schwede T, Sali A, Honig B, Levitt M, Berman HM, et al. (2009) Outcome of a workshop on applications of protein models in biomedical research. Structure 17: 151–159.
[35]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
[36]
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, et al. (2000) The Protein Data Bank. Nucleic Acids Res 28: 235–242.
[37]
Pittman YR, Valente L, Jeppesen MG, Andersen GR, Patel S, et al. (2006) Mg2+ and a key lysine modulate exchange activity of eukaryotic translation elongation factor 1B alpha. J Biol Chem 281: 19457–19468.
[38]
Andersen GR, Pedersen L, Valente L, Chatterjee I, Kinzy TG, et al. (2000) Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha. Mol Cell 6: 1261–1266.
[39]
Andersen GR, Valente L, Pedersen L, Kinzy TG, Nyborg J (2001) Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex. Nat Struct Biol 8: 531–534.
[40]
Baker D, Sali A (2001) Protein structure prediction and structural genomics. Science 294: 93–96.
[41]
Ginalski K (2006) Comparative modeling for protein structure prediction. Curr Opin Struct Biol 16: 172–177.
[42]
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
[43]
McGuffin LJ, Bryson K, Jones DT (2000) The PSIPRED protein structure prediction server. Bioinformatics 16: 404–405.
[44]
Frishman D, Argos P (1995) Knowledge-based protein secondary structure assignment. Proteins 23: 566–579.
[45]
Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234: 779–815.
[46]
Bower MJ, Cohen FE, Dunbrack RL Jr (1997) Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool. J Mol Biol 267: 1268–1282.
[47]
Canutescu AA, Shelenkov AA, Dunbrack RL Jr (2003) A graph-theory algorithm for rapid protein side-chain prediction. Protein Sci 12: 2001–2014.
[48]
Clark M, Cramer RD, Vanopdenbosch N (1989) Validation of the General-Purpose Tripos 5.2 Force-Field. Journal of Computational Chemistry 10: 982–1012.
[49]
Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck - a Program to Check the Stereochemical Quality of Protein Structures. Journal of Applied Crystallography 26: 283–291.
[50]
Vriend G (1990) What If - a Molecular Modeling and Drug Design Program. Journal of Molecular Graphics 8: 52–56.
[51]
Vriend G, Sander C (1993) Quality-Control of Protein Models - Directional Atomic Contact Analysis. Journal of Applied Crystallography 26: 47–60.
[52]
Pawlowski M, Gajda MJ, Matlak R, Bujnicki JM (2008) MetaMQAP: a meta-server for the quality assessment of protein models. BMC Bioinformatics 9: 403.
[53]
Tina KG, Bhadra R, Srinivasan N (2007) PIC: Protein Interactions Calculator. Nucleic Acids Res 35: W473–476.
[54]
Shatsky M, Nussinov R, Wolfson HJ (2004) A method for simultaneous alignment of multiple protein structures. Proteins-Structure Function and Bioinformatics 56: 143–156.
[55]
Fraczkiewicz R, Braun W (1998) Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. Journal of Computational Chemistry 19: 319–333.
[56]
Nicholls A, Sharp KA, Honig B (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281–296.
[57]
Heiden W, Moeckel G, Brickmann J (1993) A new approach to analysis and display of local lipophilicity/hydrophilicity mapped on molecular surfaces. J Comput Aided Mol Des 7: 503–514.
[58]
Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of Molecular Biology 294: 1351–1362.
[59]
Morris AL, MacArthur MW, Hutchinson EG, Thornton JM (1992) Stereochemical quality of protein structure coordinates. Proteins 12: 345–364.
[60]
Zemla A, Venclovas , Moult J, Fidelis K (2001) Processing and evaluation of predictions in CASP4. Proteins Suppl 5: 13–21.
[61]
Vitagliano L, Masullo M, Sica F, Zagari A, Bocchini V (2001) The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with GDP reveals novel features in nucleotide binding and exchange. Embo J 20: 5305–5311.
[62]
Vitagliano L, Ruggiero A, Masullo M, Cantiello P, Arcari P, et al. (2004) The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with magnesium and GDP. Biochemistry 43: 6630–6636.
[63]
Kanibolotsky DS, Novosyl'na OV, Abbott CM, Negrutskii BS, El'skaya AV (2008) 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. BMC Struct Biol 8: 4.
[64]
Marco E, Martin-Santamaria S, Cuevas C, Gago F (2004) Structural basis for the binding of didemnins to human elongation factor eEF1A and rationale for the potent antitumor activity of these marine natural products. J Med Chem 47: 4439–4452.
[65]
Pizzuti A, Gennarelli M, Novelli G, Colosimo A, Lo Cicero S, et al. (1993) Human elongation factor EF-1 beta: cloning and characterization of the EF1 beta 5a gene and assignment of EF-1 beta isoforms to chromosomes 2,5,15 and X. Biochem Biophys Res Commun 197: 154–162.
[66]
Chambers DM, Rouleau GA, Abbott CM (2001) Comparative genomic analysis of genes encoding translation elongation factor 1B(alpha) in human and mouse shows EEF1B1 to be a recent retrotransposition event. Genomics 77: 145–148.
[67]
Chang R, Wang E (2007) Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress. J Cell Biochem 100: 267–278.
[68]
Carr-Schmid A, Durko N, Cavallius J, Merrick WC, Kinzy TG (1999) Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange. J Biol Chem 274: 30297–30302.
[69]
Liu G, Tang J, Edmonds BT, Murray J, Levin S, et al. (1996) F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction. J Cell Biol 135: 953–963.
[70]
Gross SR, Kinzy TG (2005) Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology. Nat Struct Mol Biol 12: 772–778.
[71]
Gross SR, Kinzy TG (2007) Improper organization of the actin cytoskeleton affects protein synthesis at initiation. Mol Cell Biol 27: 1974–1989.
[72]
Krych M, Clemenza L, Howdeshell D, Hauhart R, Hourcade D, et al. (1994) Analysis of the functional domains of complement receptor type 1 (C3b/C4b receptor; CD35) by substitution mutagenesis. J Biol Chem 269: 13273–13278.
[73]
Krych M, Hauhart R, Atkinson JP (1998) Structure-function analysis of the active sites of complement receptor type 1. J Biol Chem 273: 8623–8629.
[74]
Iakoucheva LM, Radivojac P, Brown CJ, O'Connor TR, Sikes JG, et al. (2004) The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res 32: 1037–1049.
[75]
Kassenbrock CK, Anderson SM (2004) Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive activation by tyrosine to glutamate point mutations. J Biol Chem 279: 28017–28027.
[76]
Mayo LD, Seo YR, Jackson MW, Smith ML, Rivera Guzman J, et al. (2005) Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified. J Biol Chem 280: 25953–25959.
[77]
Sandbaken MG, Culbertson MR (1988) Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae. Genetics 120: 923–934.
[78]
Ozturk SB, Kinzy TG (2008) Guanine nucleotide exchange factor independence of the G-protein eEF1A through novel mutant forms and biochemical properties. J Biol Chem 283: 23244–23253.
[79]
Ozturk SB, Vishnu MR, Olarewaju O, Starita LM, Masison DC, et al. (2006) Unique classes of mutations in the Saccharomyces cerevisiae G-protein translation elongation factor 1A suppress the requirement for guanine nucleotide exchange. Genetics 174: 651–663.
