Cotranslational interaction of human EBP50 and ezrin overcomes masked binding site during complex assembly

Abstract

Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to “buried” domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond “simple” generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.

Multimodal cotranslational interactions direct assembly of the human multi-tRNA synthetase complex

“…2 B ). Peptide epitope mimetics (PEM) mimicking the N terminus of nascent proteins can abrogate cotranslational events ( 13 ). Moreover, the N-terminal 36 amino acids of AIMP2 are critical for its interaction with KARS1 ( Fig.…”

“…Cotranslation targeting the N terminus of an emerging peptide to form a protein complex, as observed here, presents multiple advantages. A nascent, unfolded peptide can provide recognition sites that are masked in the mature, folded protein ( 13 ). Also, the frequent presence of a “translational ramp” in which rare codons in the first ∼50 N-terminal codons hinders translation and might facilitate protein folding ( 53 ).…”

“…Cotranslational assembly offers multiple advantages, including shielding of a constituent that in free form is susceptible to aggregation or degradation or is deleterious to cell health ( 9 – 12 ). Also, the mechanism can facilitate formation of interactions between proteins in which a binding domain is masked or otherwise inaccessible in one of the mature partners ( 13 ). Protein folding during de novo synthesis is facilitated by chaperones that bind nascent protein and reduce nonspecific hydrophobic interactions; likewise, a similar role for chaperones has been proposed to facilitate cotranslational complex assembly, but experimental evidence is lacking ( 11 ).…”

Ezrin expression in female reproductive tissues: A review of regulation and pathophysiological implications

“…Ezrin phosphorylation sites can be regulated by several signaling pathways and perform different physiological functions upon activation and phosphorylation ( Yin and Schnoor, 2022 ). Initial studies demonstrated that the critical T567 on C-ERMAD could be phosphorylated by Rho-kinase (ROCK) ( Ding et al, 2022 ), STE20-like protein kinase (SLK) ( Garland et al, 2021 ), lymphocyte-oriented kinase (LOK) ( Senju and Tsai, 2022 ) and protein kinase C(PKC) ( Khan et al, 2022 ) both in the laboratory and in living organisms. Ezrin Y145, Y353 and Y477 can be modified by phosphorylation through the action of Src kinases and the intrinsic Tyr kinase activity of receptors for epidermal growth factor, hepatocyte growth factor and Platelet-derived growth factor ( Huang et al, 2018 ; Derouiche and Geiger, 2019 ; Rainey et al, 2020 ).…”

Benefits of co‐translational complex assembly for cellular fitness

“…In the canonical co-translational mechanism, a mature, fully formed protein interacts with its partner, generally near the nascent N-terminal polypeptide, as it emerges from the exit tunnel of the ribosome. [1,2] In exceptions to this mechanism, both interacting polypeptides can be mRNA-bound, that is, in-cis, in which polypeptides emerging from the same mRNA interact, or in-trans where interacting polypeptides emerge from distinct translating mRNAs. Global investigations of complex assembly suggest a widespread process where 12 of 31 and 9 of 12 complexes in fission and budding yeast, respectively, appear to be assembled cotranslationally.…”