Transmembrane but not soluble helices fold inside the ribosome tunnel

Abstract

Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.

Fig. 1

Helices in the ribosome exit tunnel. a The model protein used in this study (E. coli Lep) has two TM segments (gray) and a large C-terminal domain. Ribosome-bound truncated nascent chains of different lengths are generated by in vitro translation, in the presence of dog pancreas microsomes, of mRNAs lacking a stop codon (brown). The minimum number of residues required to span the distance between the ribosomal P-site and the active site of the OST (d, distance P-NST) will depend on the compactness of the polypeptide region located inside the ribosome tunnel. Ribosome cartoon is not drawn to scale with respect to the length of the nascent polypeptide chain nor to the membrane thickness. b In vitro translation in the absence (−) and presence (+) of rough dog pancreas microsomes (RM) of truncated mRNAs of different lengths harboring the sequences encoding different helices: VSV-G TM segment (residues 463–482), gp41 TM segment (residues 684–705), NAGK helix (residues 5–26), and L9 helix (residues 45–67). The number of residues between the Asn residue in an Asn-Ser-Thr glycosylation acceptor site and the C-terminal end of the nascent chain are shown on top. Glycosylated and non-glycosylated molecules are indicated by black and white dots, respectively. c Glycosylation profiles for constructs of the indicated lengths harboring the different helical sequences. Error bars represent the mean ± SD; n ≥ 3. Source data are provided as a Source Data file

Fig. 2

Solvent accessible surface area (SASA) for folded versus extended states. Effect of ribosome on solvent accessible surface area (SASA) of hydrophobic residues within the α-helical sequences for folded versus extended states. ΔΔSASA = ΔSASA_folded – ΔSASA_extended (see Methods for the definition of ΔSASA). Negative ΔΔSASA values indicate that the ribosome is stabilizing hydrophobic regions of the helical sequence in the folded, α-helical (compact) state more than in the extended state. Conversely, positive values indicate that the ribosome is stabilizing hydrophobic regions of the helical sequence in the extended state more than in the α-helical state

Fig. 3

TM helices from different origins display a compact conformation. Glycosylation percentage of nascent polypetides with 67 (pink) or 73 (purple) residues between the acceptor Asn and the polypeptide C-terminus. Error bars represent the mean ± SD; n ≥ 3. Individual data points are shown as green dots. Sequences and full glycosylation patterns can be found as Supplementary Fig. 7. Source data are provided as a Source Data file

Fig. 4

Hydrophobicity and helicity affect TM folding. In vitro translation of truncated VSV-G constructs (distance P-NST of 67) in which the central Ile pair (shown in bold) was mutated to less hydrophobic, charged (basic and acid residues shown in dark blue and red, respectively) and helix breaking residues (shown in gray). The average glycosylation percentage is plotted for each mutant. Error bars show the standard deviation of four or more independent experiments (p-values for the comparison with wild type (Ile pair): **<0.01 and ***<0.001). Individual data points are shown as green dots. Source data are provided as a Source Data file

Fig. 5

Folding depends on hydrophobic length and correlates with insertion. a In vitro translation in the absence (−) and presence (+) of dog pancreas rough microsomes (RM) of truncated mRNAs of the same length (distance P-NST 67) harboring VSV-G full length (lanes 1 and 2) or half (VSV-G TM.5, lanes 3 and 4) TM segment, or gp41 full length (lanes 5 and 6) or half (gp41 TM.5, lanes 7 and 8) TM segment. Glycosylated and non-glycosylated molecules are indicated by black and white dots, respectively. Amino acid sequences included are shown on top. b Schematic of the engineered leader peptidase (Lep) model protein. Lep, consisting of 2 TM segments (H1 and H2) and a large luminal domain (P2), inserts into RMs in an N_lum-C_lum orientation. In vitro protein translation in the presence (+) or absence (–) of rough microsomes (RM) and proteinase K (PK) of VSV-G (c) or gp41 (d) derived sequences. Non-glycosylated protein bands are indicated by a white dot; single and double glycosylated protein bands are indicated by one or two black dots, respectively. An upwards black triangle indicates small protected singly glycosylated H2/inserted fragment. A double downward black triangle indicated large doubly glycosylated H2/G1/trasnlocated/G2/P2 fragment. Source data are provided as a Source Data file