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.

Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαβ) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP–RNP interactions in general.

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