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Cotranslational folding of spectrin domains via partially structured states

Abstract

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.

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Figure 1: Three-helix bundle structure of spectrin domains.
Figure 2: Investigating cotranslational folding using the AP assay.
Figure 3: Force profiles.
Figure 4: Visualization of the R16 spectrin domain at the ribosomal tunnel exit.
Figure 5: Rigid body fit of the NMR structure of the R16 domain to the cryo-EM density map showing equivalent locations of R15 key folding residues.

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Acknowledgements

We thank A. Heuer for help preparing the figures. Supported by grants from the Swedish Cancer Foundation, the Swedish Research Council and the Knut and Alice Wallenberg Foundation (to G.v.H.); the Wellcome Trust (WT095195 to J.C.) and the European Research Council (ERC-2008-AdG 232648, to R.B.). J.C. is a Wellcome Trust Senior Research Fellow.

Author information

Authors and Affiliations

Authors

Contributions

O.B.N. and A.A.N. designed and carried out the experiments; J.J.H. and A.S. characterized the purified proteins; S.W. and R.B. were responsible for the cryo-EM experiments; A.S. wrote the manuscript; G.v.H. and J.C. conceived and planned the investigation and wrote the manuscript.

Corresponding authors

Correspondence to Gunnar von Heijne or Jane Clarke.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro translation of the spectrin constructs.

(a) Schematic representation of the constructs used in the AP assays showing the Lep leader (blue), GSGS and SGSG linker sites (purple), the variable linker (green), the SecM stall site (grey) and the terminal Lep segment (orange). Constructs for β16, R16m5, R16m6, R16o15c and non-folding R15 and R16 all resemble R15. L, reported linker length (minimum L=21 amino acids, maximum L=61 amino acids). R15R16m5 and R153ProR16 resemble R15R16. Note that the R16 force profiles obtained with the LepB leader sequence are virtually identical (see Main text Fig 3 a,b). (b) R16 [L=27] and R16 [L=37] translated in the PURE system. Full-length (FL) and arrested (A) products are indicated. FLc, full-length control, where the crucial Pro at the C-terminal end of the SecM AP is mutated to Ala; Ac, arrest control, where the crucial Pro at the end of the AP is substituted with a stop codon.

Supplementary Figure 2 Reproducibility of the data.

Data for three separate repeats of wild-type R16 without the Lep leader are shown. Points in red are the full R16 trace (see Main text Fig 3b), points in green are repeats collected using an in vitro translation kit with a different lot number and points in blue are repeats collected in a different laboratory using an in vitro translation kit with a different lot number.

Supplementary Figure 3 Supplementary cryo-EM images.

(a) Resolution determination of the final reconstruction using Fourier shell correlation (FSC) indicating an average resolution of 4.8 Å. (b) Calculation of the local resolution using resmap (Kucukelbir, A. et al. Nat Methods 11, 63-65, 2014). (c) Superposition of the spectrin R16 domain (red) and the previously determined cryo-EM structure of the ADR1a zinc-finger domain (gold) in the ribosome exit tunnel12. Notably, the location in the exit tunnel of the C-terminus of the R16 domain is 25-30 Å away from the location of the C-terminus of ADR1a, consistent with the difference in linker lengths (L = 33 vs. 25 residues) used in the two constructs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 460 kb)

Rigid body fit of the NMR structure of the R16 domain to the cryo-EM density map.

Rigid body fit of the NMR structure of the R16 domain colored according to r.m.s. deviation (blue, 0.5–1.9; white, 2–3.9; red, ≥4.0 Å) to the cryo-EM density map (Fig. 4c). (MP4 5049 kb)

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Nilsson, O., Nickson, A., Hollins, J. et al. Cotranslational folding of spectrin domains via partially structured states. Nat Struct Mol Biol 24, 221–225 (2017). https://doi.org/10.1038/nsmb.3355

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