Targeted lysine-to-arginine substitutions at methylation sites in eEF1A1 differentially modulate protein interactions and translation.

Lysine methylation has emerged as an important regulatory mechanism for the human translation elongation factor eEF1A, which is modified at five distinct lysine residues; however, the functional consequences of these modifications for translation remain largely unclear. By combining in vitro and cellular assays with targeted mutagenesis of the major methylation sites, we show that substitution of methylatable Lys79 with arginine enhances the interaction between eEF1A1 and its guanine nucleotide exchange complex, eEF1B, whereas the Lys36Arg mutation reduces both global translation and translation fidelity in human cells. Molecular dynamics simulations indicate that trimethylation of Lys79 partially weakens the eEF1A1-eEF1Bα interaction, an effect that can be counterbalanced by trimethylation of Lys318. These simulations also explain the previously reported absence of such inhibition in the eEF1A2 paralog. Mass spectrometry further reveals multiple nontranslational partners that preferentially associate with methylated or mutant eEF1A1 and confirms complete trimethylation of Lys36, Lys79, and Lys318 in differentiated tissue. Together, these findings highlight lysine methylation as an important modulator of eEF1A1, shaping its functions in translation and beyond.