Abstract
Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature 80S ribosomes for translational control. Here, we present a cryo-EM single particle analysis reconstruction of Ebp1 bound to non-translating human 80S ribosomes at a resolution range from 3.3 to ~8 Å. Ebp1 blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. H59 is defined as dynamic adaptor undergoing significant remodeling upon Ebp1 binding. Ebp1 recruits rRNA expansion segment ES27L to the tunnel exit via specific interactions with rRNA consensus sequences. The Ebp1-ribosome complex serves as a template for MetAP binding and provides insights into the structural principles for spatial coordination of co-translational events and molecular triage at the ribosomal tunnel exit.
Highlights
Human Ebp[1] is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation
To gain insights into Ebp[1] binding to the human 80S ribosome, we used cryo-electron microscopy (cryo-EM) single-particle analysis, and determined the structure of full-length Ebp[1] (p48 isoform) in complex with puromycin-treated 80S ribosomes purified from HeLa cells (Supplementary Fig. 2, Supplementary Table 1)
Lower local resolution for the 40S ribosomal subunit (Supplementary Fig. 3d) indicated conformational heterogeneity originating from ribosomal intersubunit rotation, which we compensated for by separating the Ebp1–ribosome complex into two independently refined segments (“2-body” approach), comprising the 40S ribosomal subunit and the 60S ribosomal subunit plus Ebp[1] and rRNA ES27L, respectively
Summary
Human Ebp[1] is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp[1] blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. It has attracted considerable attention due to its regulative role in cancer progression, the question of being “friend or foe”[1] is still unanswered This uncertainty is based on the contradictory functions of the two splice variants p42 and p48, with p42 (lacking 54 N-terminal residues) acting as tumor suppressor and p48 promoting cell proliferation[1]. Ebp[1] is MetAP-2 like and mainly distinguished from the MetAPs by the missing catalytic activity, a shorter N-terminus (~150 residues missing) and a C-terminal extension of about 50 residues harboring a highly positive charged patch including six consecutive lysine residues (Supplementary Fig. 1). C-terminal region, the lysine cluster was disordered and not part of the X-ray models (missing 30 C-terminal residues)[5,6]
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