Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

Abstract

Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome, but, despite its importance, a detailed description of such a process at the atomic level remains elusive. We show here at a residue-specific level that a nascent protein chain can reach its native tertiary structure on the ribosome. By generating translation-arrested ribosomes in which the newly synthesized polypeptide chain is selectively (13)C/(15)N-labeled, we observe, using ultrafast NMR techniques, a large number of resonances of a ribosome-bound nascent chain complex corresponding to a pair of C-terminally truncated immunoglobulin (Ig) domains. Analysis of these spectra reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain. Selective broadening of resonances for a group of residues that are colocalized in the structure demonstrates that there are specific but transient interactions between the ribosome and the N-terminal region of the folded Ig domain. These findings represent a step toward a detailed structural understanding of the cellular processes of cotranslational folding.

Fig. 1.

Schematic protocol of RNC preparation for NMR studies. (A) Structural model of the ABP-120 domains 5 and 6 [Upper, residues 644–857 of Ig2; Protein Data Bank ID code 1QFH (22)] and the construct used for the Ig2 RNC (Lower). The N-terminal domain (NTD, domain 5) and the C-terminal domain (CTD, domain 6) are designated Ig2 NTD and Ig2 CTD, respectively. The DNA plasmid of the Ig2 was digested at the unique BstNI restriction site, leading to a truncated translation product ending at residue 838, the position at which the last β-strand of the Ig2 CTD begins (highlighted in black and also indicated in the structural model). (B) Schematic description of the experimental protocol for RNC preparation (for further details, see Materials and Methods and SI Fig. 6).

Fig. 2.

¹H–¹⁵N correlation spectra of the Ig2 RNC. The SOFAST-HMQC spectra of Ig2 RNC (A), Ig2/70S (B), Ig2 RNC as in A but incubated with 1 mM puromycin (1 h, 25°C) before data acquisition (C), and isolated Ig2 NTD (red) and Ig2 (black) in the absence of 70S ribosomes (D). The cross-peaks that are absent in the Ig2 RNC spectrum (A) are indicated with red circles in A–C and are labeled with the corresponding residue identities. All spectra were recorded under identical conditions (binding buffer, 10°C) and at 700 MHz, except the spectrum in A, which was recorded at 900 MHz.

Fig. 3.

Identification of RNCs. ¹⁵N-filtered X-STE diffusion NMR measurements of Ig2 RNC (A) and purified Ig2 (B) in the presence of 70S ribosomes (Ig2/70S), recorded at 25°C, 700 MHz. Significant attenuation of the ¹⁵N-filtered signal of purified Ig2 was observed in the spectrum recorded with a strong diffusion coding/decoding gradient pair (70% Gz_max, red) flanking the 200-ms diffusion period, compared with the same spectrum recorded with a weak coding/decoding gradient pair (10% Gz_max, black) (B). The same X-STE experiments recorded for the Ig2 RNC (A) showed no discernible intensity changes, indicating the very slow diffusion of the Ig2 RNC as a result of the connection of the nascent chain to the ribosomes. (C) [³⁵S]Met radioactivity profile of sucrose gradients loaded with [³⁵S]Met-labeled Ig2 RNC. Aliquots of [³⁵S]Met-labeled Ig2 RNC were incubated at 25°C to monitor sample stability. No significant release of tRNA-bound nascent chains from the 70S ribosome was found after 8 h of incubation (red curve) vs. without incubation (black curve), whereas incubation in the presence of 1 mM puromycin over the same 8-h period induces very efficient release of the nascent chain (green curve). A time course of puromycin-induced nascent chain release under the same conditions shows that the reaction can be completed within 30 min of incubation (data not shown).

Fig. 4.

Differential line broadening in the Ig2 NTD of the Ig2 RNC. (A) Representative regions of the SOFAST-HMQC spectra of the Ig2 RNC (Left) and Ig2/70S (Right), as shown in Fig. 2 A and B, respectively. Cross-peaks that show significantly broader linewidths in the Ig2 RNC spectrum are indicated with dashed circles. The cross-peak of residue I748, which is adjacent to the domain boundary between the Ig2 NTD and CTD (Fig. 1A), cannot be identified in the Ig2 RNC spectrum (Left). (B) ¹H linewidth profiles of the Ig2 NTD in the Ig2 RNC (black open circles) and Ig2/70S (green line) obtained from the corresponding ¹H–¹⁵N SOFAST-HMQC spectra (Fig. 2 A and B). The residues in the Ig2 NTD in the Ig2 RNC whose cross-peaks exhibit severe line broadening are indicated by filled red circles, with those that exhibit linewidths >1 standard deviation above the average (dashed horizontal line) indicated by filled orange circles. Residues that are part of the β-strands of the Ig2 NTD are indicated by black arrows.

Fig. 5.

Structural mapping of the locations of the Ig2 RNC that experience NMR line broadening. (A) Schematic representation of Ig2 RNC showing relative sizes (approximate) of the 70S ribosome and the Ig2 NTD (boxed), with the loop regions in the N-terminal hemisphere that appear to interact with the ribosome indicated in red. (B) Mapping of the residues whose resonances are broadened on the structure of Ig2 NTD, depicted in ribbon representation [Protein Data Bank ID code 1QFH (22)]. The heavy atoms of those residues whose resonances are broadened by >1 standard deviation from the average (filled orange circles in Fig. 2B) and those that are broadened beyond detection in the Ig2 RNC spectra (filled red circles in Fig. 2B) are shown as orange and red spheres, respectively, and labeled with their residue identities. Residue I748, which lies at the domain boundary and whose cross-peak is absent in the Ig2 RNC spectrum (Fig. 4A), is shown by green spheres. The figure was generated by using PyMol (www.pymol.org).