Gradual compaction of the nascent peptide during cotranslational folding on the ribosome

  1. Marija Liutkute
  2. Manisankar Maiti
  3. Ekaterina Samatova
  4. Jörg Enderlein
  5. Marina V Rodnina

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Abstract

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain’s hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

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  1. Version published to 10.7554/elife.60895 on eLife
  2. eLife

    ###Reviewer #3:

    In their paper, Liutkute et al., use an elegant combination of force profile analysis (FPA) and photo-electron transfer (PET) experiments to probe the co-translational folding pathway of the N-terminal domain of the protein HemK. Over the past decades, it became increasingly clear that co-translational folding pursues different routes than those found in solution. Despite the fact that many proteins fold and unfolded many times during their lifespan after being released from the ribosome, the question of whether and how proteins fold during the process of translation is not only fundamental but also extremely difficult to access experimentally. Here, Liutkute et al. present a synergistic combination of two largely different methods to answer this question. By stalling a nascent polypeptide chain at different sequence positions and measuring the amount of full-length relative to arrested protein in a gel assay, the authors identified a sequential folding path in which the order of helix formation of the 5-helix NTD of HemK follows the order from N- to C-terminus. The authors interpret these results using the foldon concept from the Englander lab. Though the FPA is a rather qualitative experimental tool that measures the amount of molecules that crossed a certain force threshold, the analysis is striking. These experiments were complemented by PET-FCS experiments that were used to quantify the kinetic rates of conformational fluctuations of ribosome-stalled states of the protein. The conclusions drawn by the authors are that conformational fluctuations slow-down the further a protein is away from the ribosome exit tunnel. In my opinion, the work is a substantial step towards understanding the process of co-translational folding. The experiments are beautiful, well described, and the results are of clear interest to a broad readership.

    1. I would like to emphasize that care has to be taken when deducing the order of events from single time-point experiments such as FPA. The speed of translation compared to the folding speed is an important factor that eventually dictates the order at which certain structural elements will form. I admit, however, that the formation of helices, at least in solution, typically exceeds translation speeds by far, thus indicating that the identified intermediates will also form under conditions of continuous translation. Nevertheless, it would have been interesting if the authors could provide data or relevant publications about the folding speed of the HemK-NTD.

    2. The PET-FCS is indeed very appealing, however, I had some problems in understanding the actual procedure that was used for fitting. On p. 25, it is mentioned that the diffusion and triplet component based on the empirical fit with eq. 1 were subtracted from the data. Equation 1 would rather indicate that a separation of the dynamic components requires a division of the data by the relevant diffusion and triplet terms.

    3. I would call eq. 1 'empirical' rather than 'analytical'.

    4. On p. 25, the authors explain that the dynamic components of the FCS-curves were fitted using a sum of terms, one for each species. It would have been more explanatory if the authors would provide the actual equations that had been used for fitting. I would have guessed that the authors derive expressions for the correlation functions of the individual models, e.g., using the approach of Gopich & Szabo (see Eq. 1 in Gopich et al. (2009) J Chem Phys, 131, 095102), but the approach described in the methods sounds different.

    5. I was surprised that the two-step model can even provide negative, i.e., rising, amplitudes, which is very unusual for autocorrelation functions. This feature implies that the kinetic models have amplitudes that are decoupled from the actual kinetic rates. It would be great if the authors could clarify this point.

    6. I find the calculation of free energy barriers a bit overstretched given the complexity of the system. First, the pre-exponential factor of the Eyring equation (eq. 2) is only adequate for gas-phase reactions, particularly when assuming a transmission coefficient of 1. The appropriate pendant is Kramers equation. Clearly, the problem of defining the pre-exponential factor for folding reactions remains also with the Kramers expression. However, a large body of work has been dedicated to this problem over the past 20 years. It seems that a value of 1 μs-1 seems to be a good guess (see e.g. Schuler & Eaton (2008) Curr Opin Struct Biol, 18, 16). Clearly, there is no way to decide whether conformational fluctuations slow-down due to a decrease of the free energy barrier or due to a change in the pre-exponential factor.

  3. eLife

    ###Reviewer #2:

    Liutkute and coworkers use a combination of arrest peptide assays and fluorescence correlation spectroscopy to investigate the folding of the HemK N-terminal domain. Previous work from the same group has shown that the domain rapidly forms compact structures co-translationally while still partially within the ribosome exit tunnel, limited by the rate of elongation. Data from the arrest peptide assay presented here suggest that, surprisingly, stably folded structures form as soon as the first of five helices in the domain has moved past the tunnel constriction. Several additional apparent folding events occur at longer chain lengths, suggesting discrete events of structure formation within the tunnel and near its vestibule. Experiments with a destabilized mutant (4xA) indicate that some of the folding events are dependent on formation of the hydrophobic core of the domain, suggesting that they depend on tertiary structure formation. PET-FCS experiments with HemK nascent chains reveal two interconverting states, compact (C) and dynamic (D). Both states are populated similarly regardless of chain length. However, the barrier between these states increases when the domain emerges from the ribosome. These experiments indicate a destabilizing effect of the ribosome on the nascent chain. Taken together, the experiments support earlier work that proposed a sequential co-translational folding mechanism for the HemK NTD, and provide rate constants for the dynamics at the earliest stages of nascent chain folding.

    The experiments appear very carefully designed and executed, and the data is of high quality. The PET-FCS measurements in particular provide valuable quantitative information about early nascent chain folding and should be of broad interest. While the results from arrest peptide experiments are intriguing, I have concerns about their interpretation, detailed below.

    Main point:

    The arrest peptide data is interpreted entirely in terms of a pulling force on the nascent chain, generated by folding. The conclusion that formation of just one (peak I) or two (peak II) alpha-helices inside the tunnel generate substantial mechanical forces is surprising, particularly given the presumed mechanism of arrest released mediated by force. How would a force be generated by a single alpha helix? It is easier to rationalize that forces acting on the arrest peptide are generated by stable tertiary structures. However, in that case, the 4xA mutant should show much lower arrest release in the region where full folding of the domain is expected (regions VII and VIII in Fig. 1), because the mutant is largely unfolded (see Holtkamp et al., Science 2015). This effect is not observed. Together, these considerations make we wonder whether alternative explanations for the observed release rates can be ruled out. For instance, could sequence-specific effects that are not related to folding of HemK, such as local interactions of the nascent peptide with the tunnel, cause the observed changes in arrest release rates? Alternatively, could local structure formation (of an alpha helix) in the tunnel cause arrest release that is not mediated by a pulling force?

    At a minimum, the authors should discuss how they envision single alpha helices to generate the forces necessary to accelerate arrest release (which have been estimated in the literature, e.g. in Goldman et al, Science 2015, and Kemp et al., PNAS 2020).

    In addition, two control experiments should be carried out: (1) An experiment demonstrating that a bona fide unstructured protein yields more or less constant arrest release rates over a range on nascent chain lengths. Perhaps a construct starting residue 73 of HemK could serve as a control. (2) An experiment with previously characterized folded domains (e.g. some of the spectrin constructs from Kemp et al, PNAS 2020; or some of the constructs from Farías-Rico e al., PNAS 2018) to establish the fraction of full length protein (f_FL) obtained with stably folded domains under the experimental conditions used in the present manuscript. How do the f_FL values for the HemK NTD compare to fully folded proteins under the conditions used here?

  4. eLife

    ###Reviewer #1:

    This study by Liutkute et al. investigates the co-translational folding of a small alpha-helical domain from HemK. The study continues earlier studies by Rodnina and colleagues that showed using FRET and other measurements that HemK begins folding inside the ribosome exit tunnel and occurs sequentially as individual alpha-helical segments are able to be accommodated in the exit tunnel vestibule. Folding completes just outside the ribosome when the entire HemK domain is exposed. The current work extends these earlier studies using biochemical assays of "force" on the nascent chain and spectroscopic assays of intramolecular dynamics with an N-terminal fluorescent probe.

    The force assays illustrate that tension is seen as individual alpha helices move beyond the exit tunnel constriction, and at other previously documented steps of folding in the vestibule. These intra-ribosomal events are not impacted by a mutation that disrupts packing of the hydrophobic core. The fluorescence quenching dynamics show that the N-terminus is more dynamic inside the exit tunnel prior to folding and not dynamic after folding outside the tunnel. A detailed kinetic model of the fluorescence correlation data is provided to help explain the observations.

    Overall, the study provides a finer resolution view of the sequential co-translational folding of HemK. Although the broad concepts from the earlier studies are not changed by the current work, the study introduces analytical tools based on fluorescence quenching and FCS that may be useful to study the co-translational folding of other proteins.

    My primary suggestion is that the authors should be more explicit about what is being measured in the "force" sensor assay. SecM stalling relies on a specific secondary structure of the stalling sequence that causes an altered P site geometry that is unfavourable for peptide bond formation. Stalling will not occur if this altered geometry cannot be stabilized. Thus, what the authors refer to as 'force' is actually a constraint applied to the nascent chain to prevent SecM secondary structure formation. Thus, the folding is not generating force so much as constraining the nascent chain as a consequence of the ribosome exit tunnel geometry. It is a subtle, but I feel important, distinction to explain the assay. The reason is that such a constraint can actually be due to reasons other than folding. For example, an interaction between the nascent chain and the exit tunnel (or other proteins) could similarly constrain the nascent chain.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    This manuscript is in revision at eLife.

  6. Version published to 10.1101/2020.07.03.185876v1 on bioRxiv