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. 2018 Nov 27;115(48):E11284-E11293.
doi: 10.1073/pnas.1810523115. Epub 2018 Nov 9.

Folding pathway of an Ig domain is conserved on and off the ribosome

Pengfei Tian  1 , Annette Steward  2 , Renuka Kudva  3 , Ting Su  4   5 , Patrick J Shilling  3 , Adrian A Nickson  2 , Jeffrey J Hollins  2 , Roland Beckmann  4   5 , Gunnar von Heijne  6   7 , Jane Clarke  8 , Robert B Best  9
Affiliations

Folding pathway of an Ig domain is conserved on and off the ribosome

Pengfei Tian et al. Proc Natl Acad Sci U S A. .

Abstract

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-β Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Keywords: arrest peptide; fraction folded; kinetic model; mechanical force; molecular simulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cotranslational folding of the titin I27 domain by force-profile analysis. (A) The force-measurement assay. Modified with permission from ref. . I27, preceded by a His-tag, is placed L residues away from the last amino acid of the SecM AP, which in turn is followed by a 23-residue C-terminal tail derived from E. coli LepB. Constructs are translated for 15 min in the PURE in vitro translation system, and the relative amounts of arrested and full-length peptide chains produced are determined by SDS/PAGE. The fraction full-length protein, fFL, reflects the force exerted on the AP by the folding of I27 at linker length L. At short linker lengths (Top) there is not enough room in the exit tunnel for I27 to fold, little force is exerted on the AP, and the ribosome stalls efficiently on the AP (fFL ≈ 0). At intermediate linker lengths (Middle) there is enough room for I27 to fold but only if the linker segment is stretched, force is exerted on the AP, and stalling is reduced (fFL > 0). At long linker lengths (Bottom) I27 has already folded when the ribosome reaches the last codon in the AP, and again little force is exerted on the AP (fFL ≈ 0). (B) Force profiles for the I27 domain (solid squares) and the nonfolding (nf) mutant I27[W34E] (open squares). The standard error of fFL is calculated for values of L where three or more experiments were performed.
Fig. 2.
Fig. 2.
Cryo-EM structure of I27[L = 35] RNCs. (A) Cryo-EM reconstruction of the I27–TnaC[L = 35] RNC. The ribosomal small subunit is shown in yellow, the large subunit in gray, the peptidyl-tRNA with the nascent chain in green, and an additional density corresponding to I27 at the ribosome tunnel exit in red. The black cartoon eye and dashed lines indicate the angle of view in C. The density contour level for feature visualization is at 1.7 times rmsd. (B) Rigid-body fit of the I27 domain (PDB ID code 1TIT) to the cryo-EM density map displaying from high (Left) to low (Right) contour levels at 2.6, 2.0, and 1.4 rmsd, respectively. N and C represent the N and C termini of the I27 domain, respectively. (C) View looking into the exit tunnel (arrow) with density for the nascent chain (nc) in dark green. Ribosomal proteins uL29 (blue; PDB ID code 4UY8), uL24 (light green; the β hairpin close to I27 domain was remodeled based on PDB ID code 5NWY) and the fitted I27 domain (red) are shown in cartoon mode; 23S RNA and proteins not contacting I27 are shown as density only. The density contour level is at 5 rmsd excluding tRNA, nascent chain, and I27 domain, which are displayed at 1.7 rmsd.
Fig. 3.
Fig. 3.
MD simulations of cotranslational folding of I27. (A) The 50S subunit of the E. coli ribosome (PDB ID code 3OFR) with I27[L = 35] attached via an unstructured linker. (B) Coarse-grained model for I27 (red) and linker (green), with surrounding ribosomal pseudoatoms in blue. Pseudoatoms colored gray are not used in the simulations. The instantaneous force exerted on the AP is calculated from the variation in the distance x between the C-terminal Pro pseudoatom and the next pseudoatom in the linker (Inset). (C) Average forces exerted on the AP by the unfolded state (Fu, empty symbols) and folded state (Ff, filled symbols) of I27 at different linker lengths L. The average fraction folded I27 for different L,Pf, is shown in cyan on the right axis. Free energy profiles at each linker length are shown in SI Appendix, Fig. S11. (D) Experimental (red square) force profiles for cotranslational folding of I27. Force profiles calculated from simulations using the full kinetic scheme or preequilibrium model are shown by blue circles and cyan triangles, respectively. The rmsd of the fFL between experiment and simulation is 0.08.
Fig. 4.
Fig. 4.
Simulations capture the experimental force profiles for mutant I27 domains. (A) Mutated residues in I27 (sticks). (B–D) Experimental (red) force profiles and calculated ones from the full kinetic scheme (blue) for (B) I27[L58A], (C) A-strand deletion mutant I27[−A], and (D) I27[M67A]. I27[M67A] (Sim HP) represents a simulation in which hydrophobic interactions between I27[M67A] and ribosome proteins uL23/uL29 are included. Experimental force profiles for nonfolding mutants that contain an additional W34E mutation are shown as open red squares. The rmsd of the fFL between experiment and simulation for I27[L58A] and I27[−A] are 0.07 and 0.08, respectively. For I27[M67A], the fFL rmsd is 0.07 between experiment and simulation (Sim HP).
Fig. 5.
Fig. 5.
Simulated folding pathways for ribosome-tethered I27 (Left, L = 51; Middle, L = 35; Right, L = 31). (A–C) Simulated ϕ-values for I27 (black). ϕ-Values determined by in vitro folding of purified I27 are shown as red squares. At L = 51 the simulated ϕ-values match well with experiment (Spearman correlation coefficient r = 0.80). At L = 35 and L = 31 the simulated ϕ-values are higher at the N terminus and lower at the C terminus than the experimental values, reflecting a change in importance of these regions when I27 folds in the confines of the ribosome. (D–F) Relative probability that if a particular contact is formed then the protein is on a folding trajectory, p(TP|qij)nn. When the protein is constrained the limiting factor is formation of a few key contacts. A cartoon of the ribosome with I27 in red is shown in each panel. (G–I) The top 10 most important contacts are colored in cyan on the native structure.
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