Mapping Protein–Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome–Nascent Globin Complex

Abstract

Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein−ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.

Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel

“…2 . Interestingly, the emerging scenario bears a close resemblance to the structure-based “lazy lollipop” model recently developed for the partially compact RNC of a related globin 50 . This work was based on a combination of single-particle cryo-electron microscopy and fluorescence anisotropy decays 50 .…”

“…RNC crosslinking was verified by low-pH SDS-PAGE 73 and crosslinked r-protein identity was assessed by Western blotting in the absence and presence of the trigger factor (TF) chaperone. Notably, EDC leads to unequivocal identification of RNC/r-protein interactions 50 , yet it tends to underestimate interacting fractions 49 . Nonetheless, in the presence of appropriate controls, it is an extremely valuable tool to detect the existence of protein–protein interactions within the ribosome-nascent-chain complex.…”

“…The solvent-exposed nonpolar patch of the L23 r-protein, highlighted in Fig. 3 c,d, is known to interact with another nascent globin 85 . It is therefore possible that L23 establishes contacts with nonpolar regions of RNCs.…”

“…As summarized in Table 1 , these interactions were identified in a variety of experimental studies and can be divided into three categories. Namely, (i) interactions between the ribosome and nascent chains carrying an N-terminal signal sequence 18 , 38 – 42 , (ii) interactions between the ribosome and nascent chains bearing a C-terminal ribosome-stalling or arrest sequence 38 , 43 – 48 , and (iii) interactions between the ribosome and nascent chains bearing no N- or C-terminal tags 26 , 49 , 50 . Additional studies are consistent with the presence of ribosome-nascent-chain interactions, though they do not directly prove their existence 51 – 54 .…”

Correlative Super-resolution Optical and Electron Microscopic Imaging of Intracellular Ribosomal RNA by a Terpyridine Iridium(III) Complex

“…The availability of ribosomes stands as a critical determinant in a cell’s capability to regenerate proteins, thereby commanding the fundamental processes of cell growth and proliferation with marked influence . rRNA is a key component of the cellular machinery and an attractive yet unexplored target for drug design .…”