Structural Cues for Understanding eEF1A2 Moonlighting

Abstract

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIβ. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the "canonical" one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.

On the Need to Tell Apart Fraternal Twins eEF1A1 and eEF1A2, and Their Respective Outfits

“…Are G Proteins with Three Distinct Domains and Very Different Conformations in Their Monomeric and Dimeric Forms eEF1A1 and eEF1A2 fold into three well-characterized structural domains, namely, domain I (residues 4-234), where GDP/GTP binding takes place, and domains II (241-328) and III (337-462/463), both of which are β-barrels. In the monomeric 'GTP conformation' of eEF1A1 [57], Asp110 is involved in a strong ionic interaction with Arg240, but this latter residue is fully exposed to the solvent when two GDP-bound eEF1A2 monomers are mutually associated, as found in the eEF1A2 dimer from rabbit skeletal muscle solved by X-ray crystallography [58,59]. In fact, in this dimeric form, each Arg240 is located in the first half of a proline-rich motif of sequence 235 ILPPTRPTDKPLRLPL 250 that is not fully extended but has been proposed to play a critical role in sequence recognition by the SRC homology 3 (SH3) domain of effectors such as phospholipase Cγ1 (PLCγ1) and adaptors such as Nck1 and Nck2 [59].…”

“…In the monomeric 'GTP conformation' of eEF1A1 [57], Asp110 is involved in a strong ionic interaction with Arg240, but this latter residue is fully exposed to the solvent when two GDP-bound eEF1A2 monomers are mutually associated, as found in the eEF1A2 dimer from rabbit skeletal muscle solved by X-ray crystallography [58,59]. In fact, in this dimeric form, each Arg240 is located in the first half of a proline-rich motif of sequence 235 ILPPTRPTDKPLRLPL 250 that is not fully extended but has been proposed to play a critical role in sequence recognition by the SRC homology 3 (SH3) domain of effectors such as phospholipase Cγ1 (PLCγ1) and adaptors such as Nck1 and Nck2 [59].…”

“…Careful examination of the monomer-monomer interface [59] revealed the presence and high relevance for dimer stabilization of Gly70, Ile71, Asp252 and Arg423 and also Tyr86 and Lys100, which are spatially close to Asp91 and Phe98. This profusion of amino acids that are not only strictly conserved throughout evolution [34], but also substituted due to missense mutations in children affected with severe neurodevelopmental disorders (Table 2), appears to support a role for the eEF1A2 dimer (Figure 2) in cellular functions that are necessarily distinct from that of delivering tRNA to the ribosome.…”

“…Furthermore, the higher-order association of two eEF1A2 dimers in the crystal lattice gives rise to a tetramer in which the negatively charged carboxylates of Glu122 and Glu124 (also found in Table 2) interact (at the dimer-dimer interface) with the positively charged side chains of Arg166 and Lys154. In addition, Glu122 and Glu124 flank Phe123, a hydrophobic residue located at the tip of a loop that has been posited [59] as key for the interaction of the membranebound eEF1A2 dimer with PI4KB, a crucial enzyme (EC 2.7.1.67) in the biosynthesis of phosphoinositides that play essential roles in signal transduction, cytokinesis, regulation of lipid and protein transport from the Golgi complex to the PM and determination and maintenance of the identity of several cell organelles [40,60]. 2 because of their known involvement in genetic disorders are colored in green and labeled in one-character notation.…”

“…A physiological pH increase promotes the GTP-dependent binding of eEF1A to aa-tRNAs, thus changing its spatial distribution and upregulating protein translation [94]. This dynamic functional switch is likely to be driven by a conformational transition brought about by what can be considered [95] a PTM by protons, i.e., an alteration in the charge of crucial amino acid side chains (e.g., Asp35, Glu45, Glu68, Asp91, His95, Asp252, Asp362 and His367 [59]). Likewise, other PTMs can be seen as a source of additional functional richness and diversity in eEF1A1 and eEF1A2 [49] that enable these ubiquitous proteins to interact with other molecular partners in other cellular processes different from translation.…”