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Co-translational Folding Trajectory of the HemK Helical Domain Evan Mercier, and Marina V. Rodnina Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00293 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018
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Biochemistry
Co-translational Folding Trajectory of the HemK Helical Domain Evan Mercier and Marina Rodnina* Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Goettingen, Germany Ribosome, translation, peptide exit tunnel, rapid kinetics, FRET, PET Supporting Information Placeholder Abstract Protein folding begins co-translationally within the restricted space of the peptide exit tunnel of the ribosome. We have already shown that the N-terminal alpha-helical domain of the universally conserved N5-glutamine methyltransferase HemK compacts within the exit tunnel and rearranges into the native fold upon emerging from the ribosome. However, the exact folding pathway of the domain remained unclear. Here we analyzed the rapid kinetics of translation and folding monitored by FRET and PET using global fitting to a model for synthesis of the 112 amino acid-long HemK fragment. Our results suggest that the cotranslational folding trajectory of HemK starts within the tunnel and passes through four kinetically distinct folding intermediates which may represent sequential docking of helices to a growing compact core. The kinetics of the process is defined entirely by translation. The results show how analysis of ensemble kinetics data can be used to dissect complex trajectories of rapid conformational rearrangements in multicomponent systems.
During translation, the newly synthesized peptide emerges through the polypeptide exit tunnel of the ribosome. The exit tunnel covers about 30-40 amino acids of the nascent peptide in an unfolded, fully-extended conformation. The width of the tunnel does not permit formation of large tertiary structure elements due to space limitations which preclude long-range interactions necessary for the cooperative folding of larger domains. However, some structures can form within the ribosome, e.g. compacted or collapsed non-native states (1, 2), α-helices (3-5), hairpins (6-8), or even small domains (9, 10). Formation of larger tertiary structure elements can then take place when the protein emerges from the ribosome (1, 11-15). Retention of compact or intermediate states within the peptide exit tunnel may represent a fundamental feature of co-translational folding acting to prevent the chain from falling into kinetic traps, such as stably misfolded non-native conformations that may form when only a part of a protein has been synthesized. The nature of these compact folding intermediates and the kinetics of protein rearrangements on the pathway to the native fold are poorly understood. Previously, we have used real-time fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) to monitor folding of the N-terminal domain of HemK on translating ribosomes in a reconstituted in vitro translation system (1). We used mRNAs coding for 112 amino acids (aa) of HemK, which includes all 73 aa of the N-terminal domain and the following 39 aa, which should allow the complete N-terminal domain to emerge from the ribosome and fold into its native-like structure. Translation was monitored by high-resolution SDS-PAGE using a fluorophore, Bodipy FL (BOF), attached to the N-terminus of the
peptide (Figure 1A). For FRET measurements during ongoing translation, we introduced BOF as donor fluorophore at position 1 and Bodipy 576/589 (BOP) at position 34 of the nascent peptide as acceptor fluorophore. FRET reports on protein folding, because in a fully extended peptide chain the expected distance between the two fluorophores is close to 100 Å and the FRET efficiency is very low, whereas in the native state positions 1 and 34 will come in close proximity (about 25 Å), which leads to high FRET. PET reports on adjustments in protein conformation monitored by quenching of BOP at position 1 by a unique Trp residue at position 6 when the two come into close contact, as they do in the native folded structure. The position of the nascent peptide within or outside the exit tunnel and the folding status were additionally probed by a thermolysin digestion assay. In order to investigate intermediates along the co-translational folding pathway, we have previously compared the fluorescence end points of time courses obtained with ribosomes translating mRNAs truncated at different lengths (1). The time courses observed during co-translational folding of HemK, however, inherently contain the fluorescence of each intermediate as amino acids are added to the growing nascent chain. To better understand the co-translational folding trajectory of HemK, here we analyzed the FRET- and PET- dependent time courses (BOP and BOF fluorescence emissions, respectively) by global fitting to a kinetic model for HemK translation using Kintek Explorer software (16). We first constructed a simple kinetic model for HemK translation, in which the nascent chain grows by one amino acid in each step at a constant rate (Figure S1A). Fitting this model to time courses of HemK translation obtained by SDS-PAGE analysis (1) revealed that accumulation of HemK occurs more slowly than predicted by a constant rate of translation (Figure S1B). Visual inspection of translation time courses (Figure 1A) provides clear evidence of translational pausing in the form of translation intermediates which accumulate and then dissipate. Comparing translation gels for different lengths of HemK suggests two major long-lived translation intermediates, one of less than 42 aa in length, and the other between 70 and 84 aa. For more precise insight into where translational pausing can occur in HemK, we investigated ribosome profiling data (17) using the GWIPS tool (18). These data indicate high ribosome occupancy and thus potential translational pausing at positions 37-38 and 77-78, consistent with the major intermediates observed in our gels. Peaks in ribosome occupancy were also evident at these positions in other data sets (19, 20) as well as a global aggregate of data from 12 independent studies (18). Pausing was included in the kinetic model of HemK translation by introducing reversible excursions to off-pathway intermediates at codons 38 and 78, which significantly improved consistency with the translation time courses (Figure 1B). Notably, the ribosome profile revealed three addi-
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tional peaks at codons 45, 59-60, and 90, which could be assigned to low-abundance intermediates in our translation time courses (Figure 1A), although modeling pauses at these sites did not improve the fit.
FIGURE 2. Observation of co-translational folding in real time. Time courses (black) were obtained by translating mRNAs encoding the indicated lengths of HemK in a stopped-flow apparatus using a purified in vitro translation system. Fluorescence changes of (A) FRET-labeled and (B,C) PET-labeled nascent HemK in the presence (B) or absence (C) of Trp quencher. The red lines were obtained by global fitting.
FIGURE 1. Kinetic model of translation. (A) Time courses of in vitro translation were analyzed by SDS-PAGE (1). Long-lived translation intermediates corresponding to HemK 38 and HemK 78 are indicated by red and yellow asterisks, respectively. (B) Kinetic model wherein the HemK nascent chain is extended one amino acid at a time at a rate of ktrans. Translational pausing is modeled by reversible excursions to non-translating states at nascent chain lengths of 38 and 78 amino acids. (C) Quantification of full-length product formation obtained from SDS-PAGE (black symbols) plotted along with the fits obtained from global fitting (red lines). The kinetic model containing a total of 114 states was then globally fit to experimentally measured translation and FRET- or PET-based co-translational folding time courses of HemK 42, 56, 70, 84, 98, and 112 (the number indicates the aa length of the protein product). The quality of the fits (Figure 2) indicates that HemK translation can be adequately described by the model in which each amino acid is added at the same rate (4.5 aa/s). This rate is slightly faster than the average rate of translation we reported earlier for these experiments (3.6 aa/s), because the average rate also reflects the effect of translational pausing. The kinetic traps at codons 38 and 78 are described by relatively fast excursions to non-translating states and slow recovery to the translation pathway (Table 1).
Table 1. Kinetic parameters of translation determined by global fitting.
ktrans /aa·s-1 -1
ktrap, 38 /s
-1
FRET
PET (+ Trp)
PET (– Trp)
4.5 ± 0.1
4.3 ± 0.1
4.5 ± 0.1
330 ± 120
320 ± 120
330 ± 140
krec, 38 /s
10 ± 3
10 ± 3
10 ± 4
ktrap, 78 /s-1
9.7 ± 2.0
7.3 ± 2.3
10 ± 4
krec, 78 /s-1
0.12 ± 0.03
0.08 ± 0.04
0.12 ± 0.05
In addition to kinetic parameters, global fitting also assigns Intrinsic Fluorescence Intensities (IFIs (21)) to each of the 114 HemK species in the mechanism. We have previously used IFIs derived from global fitting to characterize the motions of different elements within the ribosome during mRNA translocation (21). IFI analysis allows extraction of the minimum number of intermediates on the reaction pathway as well as the relative fluorescence of these intermediates from ensemble kinetics data. Here we compute IFIs as a means of characterizing kinetic intermediates present on the ribosome during cotranslational folding of HemK. When all 114 IFIs are freely adjustable during the fit, the resulting values are statistically undefined, which is reported in the KinTek fit results. By collecting HemK species of similar lengths into groups that share a single IFI, the number of adjustable parameters in the fit is reduced to a point where all IFIs could be statistically defined. Iterative rounds of grouping IFIs and fitting were carried out until a maximum number of IFIs could be defined within the limits of uncertainty provided by the covariance matrix derived during nonlinear regression. This approach allows estimation of the minimum number of intermediates on the folding pathway. We note that the FRET-based IFIs are not equivalent to FRET efficiencies (which would lie in the range 0 to 1), because the fitted time courses for FRET-labeled HemK were obtained from acceptor fluorescence emissions only. The resulting fits reveal 14 FRET-based IFIs and 28 PET-based IFIs which reflect different states along the co-translational folding trajectory. These states include the initiation complex as well as the IFIs of the nascent peptides HemK 42, 56, 70, 84, 98, and 112 obtainable by endpoint analysis of the fluorescence data. The global fitting approach, however, also reveals IFIs for additional intermediates between each of these states. The newly identified intermediate IFIs do not necessarily represent single intermediate conformations since equally good fits can be obtained by subdividing the grouped IFIs, although the uncertainty of the fitted parameters increases as a result. Rather, they represent an average fluorescence of an ensemble of intermediates that can be grouped to obtain a minimal defined folding trajectory. The IFIs plotted in Figure 3 represent a detailed view of the cotranslational folding pathway for the helical domain of HemK. Small increases in FRET-based IFIs are observed as the ribosome translates codons 7 and 10, before addition of the acceptor fluorophore at position 34. These states represent donor-only fluorescence increases, possibly when the N-terminus of the nascent peptide passes through the constriction of the exit tunnel formed by ribosomal proteins L4 and L22 about 30 Å away from the pep-
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Biochemistry
tidyl transferase center. The PET values increase at position 6, when the tryptophan quencher is added to the nascent chain, and again at amino acids 10-11, when the BOP label is near the constriction. When the ribosome synthesizes the polypeptide of 40-41 aa, the FRET-based IFIs increase transiently. This high-FRET state is observed when the acceptor label is located 6-7 amino acids away from the peptidyl transferase center of the ribosome. No signal change at this peptide length is observed in the donoronly or acceptor-only controls (1) suggesting that increase in these IFI values represents a very high-FRET/low-PET intermediate. This may reflect a transient hairpin-like conformation of the protein formed in the upper part of the exit tunnel and possibly relate to spatial constraints posed by the constriction. Incorporation of the next 14 aa is not reflected in FRET changes, but the PET-based IFIs increase slightly and remain at a similar level until a length of 85 amino acids (Figure 3). When the chain is extended to 56-70 amino acids, the FRET-based IFI values increase. The 70 amino acid construct was previously identified in the endpoint analysis as a compact non-native intermediate fully occluded by the exit tunnel (1). This compact non-native intermediate represents an ensemble of structures that are formed when helices 1-4 are confined within the exit tunnel. The FRET IFIs then decrease as the chain is extended from 70 to 84 amino acids, suggesting that the initial compact intermediate rearranges into a more relaxed state where the fluorescence labels are further apart or oriented in such a way that the average FRET signal is lower than in the preceding high-compaction state. This intermediate is distinct from the fully-folded domain as indicated by thermolysin digestion experiments using HemK84 (1).
FIGURE 3. Intermediates of co-translational folding. IFIs calculated from global fitting are based on FRET- (A) or PET-labeled (B) nascent HemK. PET-based IFIs were computed as the difference of (+Trp) and (–Trp) (Figure 2B,C). Vertical dotted lines indicate positions where translational pausing was incorporated into the kinetic model. Intermediates with distinct FRET or PET IFIs are indicated by shaded areas and labeled I-V, and fluorescence values determined by endpoint analysis in (1) are shown as red symbols. The global-fitting approach reveals the existence of yet another high-FRET/low-PET intermediate state as the chain is extended from 84 to 98 amino acids. Importantly, the existence of this highFRET intermediate remained hidden in endpoint analysis, because HemK 84 and 98 have similar IFIs. When the chain length
reached 98 aa, helix 4 (aa 49-63), which contributes two of four leucine residues to the hydrophobic core of HemK, emerges from the exit tunnel. Protease digestion experiments suggest that the conformation of the nascent HemK98 is close to that of natively folded protein (1). However, at a length of about 85-97, a part of helix 4 has not yet emerged because the exit tunnel occludes at least 30 aa. Therefore, the high-FRET intermediate with 85-97 aa likely reflects an intermediate formed upon inclusion of helix 4 into the hydrophobic core of HemK within the constrained space of the lower part of the exit tunnel. The FRET-based IFIs calculated for HemK 98 to HemK 112 do not change, indicating that the relative positions of the FRET labels are similar for all of these constructs; previous protease digestion experiments suggested that this final conformation is similar to the native structure of the HemK NTD in solution (1). The PET-based IFIs, however, reveal a high-PET intermediate between HemK 98 and HemK 103 which corresponds to a length where helix 5 has yet to emerge from the vestibule of the ribosome. At nascent chain lengths above 102, the PET IFIs decrease to the low-PET values observed in the native conformation. In conclusion, these results suggest that co-translational folding of HemK NTD proceeds through at least four intermediates (I, II, III, and IV in Figure 3) and is rate-limited only by translation. Furthermore, the degree of compaction at each intermediate is consistent with a linear folding trajectory where helices are added one by one to a compacted core. Within this molecular hypothesis, the speculative folding trajectory of HemK’s N-terminal domain would proceed as follows. Peptide compaction starts within the exit tunnel when helices 1-4 have been synthesized, but all of the elements are still occluded by the ribosome. This initial compact intermediate is characterized by high FRET (state I, Figure 4), which would be consistent with a roughly antiparallel arrangement of helices 1 and 2. This intermediate then rearranges into a less compact state (state II). Formation of state II occurs when the N-terminus starts to emerge from the exit tunnel, which likely reflects the additional space required for the addition of helix 3 to the compacted core. Further movement of the nascent peptide down the exit tunnel leads to further transient compaction (state III), when helix 4 has reached the lower part of the tunnel (the vestibule). This high-FRET intermediate would involve inclusion of helix 4 into the hydrophobic core, but adopt a compact conformation due to the restrictive space of the vestibule. After emergence of helix 4 from the peptide exit tunnel, the nascent chain experiences a decompaction to state IV characterized by native-like FRET, but high PET. Upon emergence of helix 5 from the vestibule, the PET decreases to native-like levels suggesting that addition of helix 5 to the folded N-terminus occurs outside the vestibule. While we feel that accretion of helices into a growing domain core is the most parsimonious interpretation of the data, we do not exclude that N-terminal portions of the nascent chain may form on-pathway native-like intermediates resembling molten globules(22), or undergo non-specific collapse of the nascent chain (23, 24) within the vestibule of the peptide exit tunnel of the ribosome. Our results demonstrate a sequence of folding intermediates which, regardless of their nature, change as the nascent chain is extended by the ribosome. Although protein folding is believed to be intrinsically heterogeneous, rapid kinetics provides a way to identify sequential co-translational protein folding intermediates as they appear in real time on (or in) the ribosome. The present analysis of the kinetic data highlights the power of ensemble rapid kinetics approaches to delineate mechanisms in complex multicomponent systems.
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polyacrylamide gel electrophoresis; IFI, intrinsic fluorescence intensities.
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ASSOCIATED CONTENT
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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details of biochemical materials, experimental conditions, and global fitting procedure. (PDF)
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected].
Funding Sources The work was supported by the Max Planck Society and a grant of the Deutsche Forschungsgemeinschaft (SFB1190).
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT The authors thank Wolfgang Wintermeyer for critical reading of the manuscript.
ABBREVIATIONS FRET, fluorescence resonance energy transfer; PET, photoinduced electron transfer; aa, amino acids; BOF, Bodipy FL; BOP, Bodipy 576/589; SDS-PAGE, sodium dodecyl sulfate–
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