Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.

CEITEC Masaryk University Brno Czech Republic

CIC bioGUNE Basque Research and Technology Alliance Derio Spain

Department of Physical Biochemistry Max Planck Institute for Biophysical Chemistry Gottingen Germany

IKERBASQUE Basque Foundation for Science Bilbao Spain

Comment In

Cell Cycle. 2022 Aug;21(16):1663-1666. doi: 10.1080/15384101.2022.2062186. PubMed

See more in PubMed

Afonine PV, Klaholz BP, Moriarty NW, Poon BK, Sobolev OV, Terwilliger TC, Adams PD, Urzhumtsev A (2018a) New tools for the analysis and validation of cryo‐EM maps and atomic models. Acta Crystallogr D Struct Biol 74: 814–840 PubMed PMC

Afonine PV, Poon BK, Read RJ, Sobolev OV, Terwilliger TC, Urzhumtsev A, Adams PD (2018b) Real‐space refinement in PHENIX for cryo‐EM and crystallography. Acta Crystallogr D Struct Biol 74: 531–544 PubMed PMC

Agirrezabala X, Samatova E, Klimova M, Zamora M, Gil‐Carton D, Rodnina MV, Valle M (2017) Ribosome rearrangements at the onset of translational bypassing. Sci Adv 3: e1700147 PubMed PMC

Balchin D, Hayer‐Hartl M, Hartl FU (2016) In vivo aspects of protein folding and quality control. Science 353: aac4354 PubMed

Baram D, Yonath A (2005) From peptide‐bond formation to cotranslational folding: dynamic, regulatory and evolutionary aspects. FEBS Lett 579: 948–954 PubMed

Bashan A, Agmon I, Zarivach R, Schluenzen F, Harms J, Berisio R, Bartels H, Franceschi F, Auerbach T, Hansen HA et al (2003) Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression. Mol Cell 11: 91–102 PubMed

Bhushan S, Gartmann M, Halic M, Armache JP, Jarasch A, Mielke T, Berninghausen O, Wilson DN, Beckmann R (2010) alpha‐Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat Struct Mol Biol 17: 313–317 PubMed

Buhr F, Jha S, Thommen M, Mittelstaet J, Kutz F, Schwalbe H, Rodnina MV, Komar AA (2016) Synonymous codons direct cotranslational folding toward different protein conformations. Mol Cell 61: 341–351 PubMed PMC

Burnley T, Palmer CM, Winn M (2017) Recent developments in the CCP‐EM software suite. Acta Crystallogr D Struct Biol 73: 469–477 PubMed PMC

Cabrita LD, Cassaignau AME, Launay HMM, Waudby CA, Wlodarski T, Camilloni C, Karyadi ME, Robertson AL, Wang X, Wentink AS et al (2016) A structural ensemble of a ribosome‐nascent chain complex during cotranslational protein folding. Nat Struct Mol Biol 23: 278–285 PubMed PMC

Cassaignau AME, Cabrita LD, Christodoulou J (2020) How does the ribosome fold the proteome? Annu Rev Biochem 89: 389–415 PubMed

Chatterjee S, Jiang W, Emerson SD, Inouye M (1993) The backbone structure of the major cold‐shock protein CS7.4 of Escherichia coli in solution includes extensive beta‐sheet structure. J Biochem 114: 663–669 PubMed

Chen E, Everett ML, Holzknecht ZE, Holzknecht RA, Lin SS, Bowles DE, Parker W (2010) Short‐lived alpha‐helical intermediates in the folding of beta‐sheet proteins. Biochemistry 49: 5609–5619 PubMed

Clark PL, King J (2001) A newly synthesized, ribosome‐bound polypeptide chain adopts conformations dissimilar from early in vitro refolding intermediates. J Biol Chem 276: 25411–25420 PubMed

Clarke TF, Clark PL (2008) Rare codons cluster. PLoS One 3: e3412 PubMed PMC

Doerfel LK, Wohlgemuth I, Kothe C, Peske F, Urlaub H, Rodnina MV (2013) EF‐P is essential for rapid synthesis of proteins containing consecutive proline residues. Science 339: 85–88 PubMed

Eichmann C, Preissler S, Riek R, Deuerling E (2010) Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy. Proc Natl Acad Sci USA 107: 9111–9116 PubMed PMC

Emsley P, Cowtan K (2004) Coot: model‐building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132 PubMed

Evans MS, Sander IM, Clark PL (2008) Cotranslational folding promotes beta‐helix formation and avoids aggregation in vivo. J Mol Biol 383: 683–692 PubMed PMC

Fu Z, Indrisiunaite G, Kaledhonkar S, Shah B, Sun M, Chen B, Grassucci RA, Ehrenberg M, Frank J (2019) The structural basis for release‐factor activation during translation termination revealed by time‐resolved cryogenic electron microscopy. Nat Commun 10: 2579 PubMed PMC

Goldman DH, Kaiser CM, Milin A, Righini M, Tinoco I, Bustamante C (2015) Mechanical force releases nascent chain‐mediated ribosome arrest in vitro and in vivo . Science 348: 457–460 PubMed PMC

Guinn EJ, Tian P, Shin M, Best RB, Marqusee S (2018) A small single‐domain protein folds through the same pathway on and off the ribosome. Proc Natl Acad Sci USA 115: 12206–12211 PubMed PMC

Holtkamp W, Kokic G, Jager M, Mittelstaet J, Komar AA, Rodnina MV (2015) Cotranslational protein folding on the ribosome monitored in real time. Science 350: 1104–1107 PubMed

Ismail N, Hedman R, Schiller N, von Heijne G (2012) A biphasic pulling force acts on transmembrane helices during translocon‐mediated membrane integration. Nat Struct Mol Biol 19: 1018–1022 PubMed PMC

Jakobi AJ, Wilmanns M, Sachse C (2017) Model‐based local density sharpening of cryo‐EM maps. Elife 6: e27131 PubMed PMC

Jin H, Kelley AC, Loakes D, Ramakrishnan V (2010) Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release. Proc Natl Acad Sci USA 107: 8593–8598 PubMed PMC

Kaiser CM, Goldman DH, Chodera JD, Tinoco I Jr, Bustamante C (2011) The ribosome modulates nascent protein folding. Science 334: 1723–1727 PubMed PMC

Kelkar DA, Khushoo A, Yang Z, Skach WR (2012) Kinetic analysis of ribosome‐bound fluorescent proteins reveals an early, stable, cotranslational folding intermediate. J Biol Chem 287: 2568–2578 PubMed PMC

Kemp G, Nilsson OB, Tian P, Best RB, von Heijne G (2020) Cotranslational folding cooperativity of contiguous domains of alpha‐spectrin. Proc Natl Acad Sci USA 117: 14119–14126 PubMed PMC

Komar AA, Jaenicke R (1995) Kinetics of translation of gamma B crystallin and its circularly permutated variant in an in vitro cell‐free system: possible relations to codon distribution and protein folding. FEBS Lett 376: 195–198 PubMed

Kosolapov A, Deutsch C (2009) Tertiary interactions within the ribosomal exit tunnel. Nat Struct Mol Biol 16: 405–411 PubMed PMC

Kucukelbir A, Sigworth FJ, Tagare HD (2014) Quantifying the local resolution of cryo‐EM density maps. Nat Methods 11: 63–65 PubMed PMC

Lim VI (1978) Polypeptide chain folding through a highly helical intermediate as a general principle of globular protein structure formation. FEBS Lett 89: 10–14 PubMed

Lim VI, Spirin AS (1986) Stereochemical analysis of ribosomal transpeptidation – Conformation of nascent peptide. J Mol Biol 188: 565–574 PubMed

Liutkute M, Maiti M, Samatova E, Enderlein J, Rodnina MV (2020a) Gradual compaction of the nascent peptide during cotranslational folding on the ribosome. Elife 9: e60895 PubMed PMC

Liutkute M, Samatova E, Rodnina MV (2020b) Cotranslational folding of proteins on the ribosome. Biomolecules 10: 97 PubMed PMC

Lu J, Deutsch C (2005) Folding zones inside the ribosomal exit tunnel. Nat Struct Mol Biol 12: 1123–1129 PubMed

Maracci C, Wohlgemuth I, Rodnina MV (2015) Activities of the peptidyl transferase center of ribosomes lacking protein L27. RNA 21: 2047–2052 PubMed PMC

Marino J, von Heijne G, Beckmann R (2016) Small protein domains fold inside the ribosome exit tunnel. FEBS Lett 590: 655–660 PubMed

Marsden AP, Hollins JJ, O'Neill C, Ryzhov P, Higson S, Mendonca C, Kwan TO, Kwa LG, Steward A, Clarke J (2018) Investigating the effect of chain connectivity on the folding of a beta‐sheet protein on and off the ribosome. J Mol Biol 430: 5207–5216 PubMed PMC

Mercier E, Rodnina MV (2018) Co‐translational folding trajectory of the HemK helical domain. Biochemistry 57: 3460–3464 PubMed

Milon P, Konevega AL, Peske F, Fabbretti A, Gualerzi CO, Rodnina MV (2007) Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria. Methods Enzymol 430: 1–30 PubMed

Mittelstaet J, Konevega AL, Rodnina MV (2013) A kinetic safety gate controlling the delivery of unnatural amino acids to the ribosome. J Am Chem Soc 135: 17031–17038 PubMed

Nakatogawa H, Ito K (2002) The ribosomal exit tunnel functions as a discriminating gate. Cell 108: 629–636 PubMed

Nilsson OB, Hedman R, Marino J, Wickles S, Bischoff L, Johansson M, Muller‐Lucks A, Trovato F, Puglisi JD, O'Brien EP et al (2015) Cotranslational protein folding inside the ribosome exit tunnel. Cell Rep 12: 1533–1540 PubMed PMC

Notari L, Martinez‐Carranza M, Farias‐Rico JA, Stenmark P, von Heijne G (2018) Cotranslational folding of a pentarepeat beta‐helix protein. J Mol Biol 430: 5196–5206 PubMed

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612 PubMed

Reid KL, Rodriguez HM, Hillier BJ, Gregoret LM (1998) Stability and folding properties of a model beta‐sheet protein, Escherichia coli CspA. Protein Sci 7: 470–479 PubMed PMC

Rodnina MV, Wintermeyer W (1995) GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs. Proc Natl Acad Sci USA 92: 1945–1949 PubMed PMC

Schagger H (2006) Tricine‐SDS‐PAGE. Nat Protoc 1: 16–22 PubMed

Schagger H, von Jagow G (1987) Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379 PubMed

Schindelin H, Jiang W, Inouye M, Heinemann U (1994) Crystal structure of CspA, the major cold shock protein of Escherichia coli. Proc Natl Acad Sci USA 91: 5119–5123 PubMed PMC

Schmeing TM, Huang KS, Strobel SA, Steitz TA (2005) An induced‐fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl‐tRNA. Nature 438: 520–524 PubMed

Su T, Cheng J, Sohmen D, Hedman R, Berninghausen O, von Heijne G, Wilson DN, Beckmann R (2017) The force‐sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling. Elife 6: e25642 PubMed PMC

Svidritskiy E, Madireddy R, Korostelev AA (2016) Structural basis for translation termination on a pseudouridylated stop codon. J Mol Biol 428: 2228–2236 PubMed PMC

Terwilliger TC, Sobolev OV, Afonine PV, Adams PD (2018) Automated map sharpening by maximization of detail and connectivity. Acta Crystallogr D Struct Biol 74: 545–559 PubMed PMC

Tian P, Steward A, Kudva R, Su T, Shilling PJ, Nickson AA, Hollins JJ, Beckmann R, von Heijne G, Clarke J et al (2018) Folding pathway of an Ig domain is conserved on and off the ribosome. Proc Natl Acad Sci USA 115: E11284–E11293 PubMed PMC

To P, Whitehead B, Tarbox HE, Fried SD (2021) Nonrefoldability is pervasive across the E. coli proteome. J Am Chem Soc 143: 11435–11448 PubMed PMC

Voorhees RM, Weixlbaumer A, Loakes D, Kelley AC, Ramakrishnan V (2009) Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome. Nat Struct Mol Biol 16: 528–533 PubMed PMC

Vu DM, Reid KL, Rodriguez HM, Gregoret LM (2001) Examination of the folding of E. coli CspA through tryptophan substitutions. Protein Sci 10: 2028–2036 PubMed PMC

Wall EA, Caufield JH, Lyons CE, Manning KA, Dokland T, Christie GE (2015) Specific N‐terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria. Mol Microbiol 95: 258–269 PubMed PMC

Walsh IM, Bowman MA, Soto Santarriaga IF, Rodriguez A, Clark PL (2020) Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness. Proc Natl Acad Sci USA 117: 3528–3534 PubMed PMC

Wilson DN, Arenz S, Beckmann R (2016) Translation regulation via nascent polypeptide‐mediated ribosome stalling. Curr Opin Struct Biol 37: 123–133 PubMed

Woolhead CA, McCormick PJ, Johnson AE (2004) Nascent membrane and secretory proteins differ in FRET‐detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116: 725–736 PubMed

Youngman EM, Brunelle JL, Kochaniak AB, Green R (2004) The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell 117: 589–599 PubMed

Zhang G, Hubalewska M, Ignatova Z (2009) Transient ribosomal attenuation coordinates protein synthesis and co‐translational folding. Nat Struct Mol Biol 16: 274–280 PubMed

Zhang K (2016) Gctf: Real‐time CTF determination and correction. J Struct Biol 193: 1–12 PubMed PMC

Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA (2017) MotionCor2: anisotropic correction of beam‐induced motion for improved cryo‐electron microscopy. Nat Methods 14: 331–332 PubMed PMC

Zivanov J, Nakane T, Forsberg BO, Kimanius D, Hagen WJ, Lindahl E, Scheres SH (2018) New tools for automated high‐resolution cryo‐EM structure determination in RELION‐3. Elife 7: e42166 PubMed PMC

See more in PubMed

PDB
7NWW, 7OIF, 7OIG, 7OT5, 7OII