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. 2020 Jan 21;117(3):1485-1495.
doi: 10.1073/pnas.1913207117. Epub 2020 Jan 7.

Cotranslational folding allows misfolding-prone proteins to circumvent deep kinetic traps

Amir Bitran  1   2 , William M Jacobs  3 , Xiadi Zhai  1 , Eugene Shakhnovich  4
Affiliations

Cotranslational folding allows misfolding-prone proteins to circumvent deep kinetic traps

Amir Bitran et al. Proc Natl Acad Sci U S A. .

Abstract

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.

Keywords: codon usage; cotranslational folding; evolution; protein folding; self-assembly.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
(Top Left) We run replica exchange atomistic simulations with a knowledge-based potential and umbrella sampling to compute a protein’s free-energy landscape. (Bottom Left) To obtain barrier heights, we run high-temperature unfolding simulations and extrapolate unfolding rates down to lower temperatures assuming Arrhenius kinetics. (Top Right) The principle of detailed balance is then used to compute folding rates. (Bottom Right) The process is repeated at multiple chain lengths and incorporated into a kinetic model of cotranslational folding. For details, see Materials and Methods.
Fig. 2.
Fig. 2.
(A) Structure of native MarR dimer bound to DNA (Left) as well as monomer (Right) with highlighted dimerization region (green), DNA-binding region (blue), and a crucial beta hairpin involved in stabilizing the DNA-binding region (gold). (B) Mean fraction of native contacts per subunit for monomeric and dimeric MarR as a function of temperature normalized by DNA-binding region melting temperature (right dotted line). The dimer melting temperature is indicated by the left dotted line. Sample monomeric structures from each temperature range are shown, illustrating melting of the dimerization region followed by the DNA-binding region. (C) Predicted folding pathway of MarR monomer. (See text for details.) (D) (Top) At various chain lengths, we plot the equilibrium probability that the structural elements associated with each folding step in the MarR monomer folding pathway are folded (gold, hairpin folding; blue, DNA-binding region folding; green, dimerization region folding). Xs indicate the minimum chain lengths at which each step is possible. (Bottom) For each chain length shown in Top, we plot the rate of the slowest folding step–DNA-binding region formation. A narrow window of chain lengths that confers both folding speed and stability is highlighted in purple. Error bars on folding rates are obtained from bootstrapping (Materials and Methods). Both Top and Bottom are shown at a simulation temperature of T=0.51TM.
Fig. 3.
Fig. 3.
(A) Folding rate vs. temperature for DNA-binding region folding rate as a function of temperature at nascent chain length 100 (dashed line) and full MarR (solid line), using the all-atom potential (Left) and a native-central potential in which nonnative interactions have been turned off (Right). Symbols indicate temperatures at which the partial chain folds significantly faster than the full monomer (p<0.01) based on bootstrapped distributions (Materials and Methods). Rates are plotted only at temperatures where the folding free-energy difference is ≤20 kBT owing to large statistical uncertainties associated with free-energy differences greater than this. The resulting temperature range is different in the two potentials, hence the differing x scales. (B) Free-energy difference between configurations prior to the rate-limiting step that are kinetically trapped (defined as having at least five nonnative contacts that must be broken before the rate-limiting step can occur) and those that are not trapped as a function of temperature for both the partial MarR chain at length 100 and full MarR. (C) Mean nonnative contact maps for the two most prevalent clusters (Materials and Methods) among full MarR simulation snapshots in which the DNA-binding region is not folded, along with representative structures. Contacts involving the C terminus that must be broken before folding can proceed are circled in red on the maps and highlighted on the respective structures.
Fig. 4.
Fig. 4.
(A) Schematic of kinetic model (see main text and Materials and Methods for details). Dimerization is shown for completeness, but not accounted for in the kinetic model. (B) Time evolution for the probability of occupying different states as a function of time, assuming the slowest folding rate is 6⋅10−3 times the protein synthesis rate (under constant translation speed). We further assume either no slowdown at conserved rare codons between residues 100 and 112 (Left) or a sixfold slowdown at rare codons (Right) (main text and Materials and Methods). States are colored as in A (black, no native tertiary structure; gold, beta hairpin folded; red, beta hairpin folded with significant nonnative contacts; blue, DNA-binding region folded; green, fully folded), and sample structures are shown. We neglect lengths prior to 100, at which point no folding occurs. (C) Fractional reduction in the mean time to complete synthesis and folding as a function of unknown synthesis rate, assuming various percent slowdowns at rare codons indicated by numbers over the curves.
Fig. 5.
Fig. 5.
(A–D) As a function of chain length, the equilibrium probability that tertiary structure elements associated with the rate-limiting step are formed (Top) and the folding rate associated with the rate-limiting step (Bottom) are shown for proteins (A) FabG, (B) CMK, (C) DHFR, and (D) HemK. For each protein, the native structure (A–D, Top) and a sample structure that has yet to undergo the rate-limiting folding step (A–D, Bottom) are shown, with C-terminal nonnative contacts that must be broken prior to this step highlighted in red. Blue Xs in A and D, Top indicate the lengths at which the first amino acids associated with the rate-limiting step have been synthesized, while black Xs in B and C, Bottom indicate that no folding rate is computed because, even though enough residues have been synthesized for the rate-limiting structures to fold, their stability is low. As before, for each protein, we work at a temperature at which the fully synthesized chain shows a folding stability of ∼5 to 15 kBT. For more details pertaining to each protein, see SI Appendix. (E) For each protein simulated, we indicate whether stable cotranslational folding intermediates are formed, deep kinetic traps slow folding, and conserved C-terminal rare codons are found in the sequence.
Fig. 6.
Fig. 6.
For misfolding-prone proteins that can fold cotranslationally, the overall folding rate is optimized if the nascent chain has time to start folding at the earliest length at which stable folding can occur. At this point, the chain’s folding landscape is still relatively smooth (blue arrow). In the case that the nascent chain’s folding rate at this critical length is slightly slower than the synthesis rate, then slowing down synthesis using rare codons roughly 30 amino acids downstream is beneficial. In contrast, delaying folding until further synthesis is complete (red arrow) leads to deep kinetic traps stabilized by C-terminal residues, which significantly slow folding.
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References

    1. Naganathan A. N., Muñoz V., Scaling of folding times with protein size. J. Am. Chem. Soc. 127, 480–481 (2005). - PubMed
    1. Houwman J. A., van Mierlo C. P., Folding of proteins with a flavodoxin-like architecture. FEBS J. 284, 3145–3167 (2017). - PubMed
    1. Suren T., et al. , Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor. Proc. Natl. Acad. Sci. U.S.A. 115, 11688–11693 (2018). - PMC - PubMed
    1. Scholl Z. N., Yang W., Marszalek P. E., Chaperones rescue luciferase folding by separating its domains. J. Biol. Chem. 289, 28607–28618 (2014). - PMC - PubMed
    1. Sohl J. L., Jaswal S. S., Agard D. A., Unfolded conformations of α-lytic protease are more stable than its native state. Nature 395, 817–819 (1998). - PubMed

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