Partially inserted nascent chain unzips the lateral gate of the Sec translocon
2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.15252/embr.201948191
ISSN1469-3178
AutoresLukas Kater, Benedikt Frieg, Otto Berninghausen, Holger Gohlke, Roland Beckmann, Alexej Kedrov,
Tópico(s)Enzyme Structure and Function
ResumoReport5 August 2019Open Access Transparent process Partially inserted nascent chain unzips the lateral gate of the Sec translocon Lukas Kater Lukas Kater Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Search for more papers by this author Benedikt Frieg Benedikt Frieg orcid.org/0000-0002-7877-0262 John von Neumann Institute for Computing, Jülich Supercomputing Centre, Institute for Complex Systems - Structural Biochemistry (ICS-6), Forschungszentrum Jülich GmbH, Jülich, Germany Search for more papers by this author Otto Berninghausen Otto Berninghausen Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Search for more papers by this author Holger Gohlke Corresponding Author Holger Gohlke [email protected] orcid.org/0000-0001-8613-1447 John von Neumann Institute for Computing, Jülich Supercomputing Centre, Institute for Complex Systems - Structural Biochemistry (ICS-6), Forschungszentrum Jülich GmbH, Jülich, Germany Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Roland Beckmann Corresponding Author Roland Beckmann [email protected] orcid.org/0000-0003-4291-3898 Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Search for more papers by this author Alexej Kedrov Corresponding Author Alexej Kedrov [email protected] orcid.org/0000-0001-9117-752X Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Synthetic Membrane Systems, Institute for Biochemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Lukas Kater Lukas Kater Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Search for more papers by this author Benedikt Frieg Benedikt Frieg orcid.org/0000-0002-7877-0262 John von Neumann Institute for Computing, Jülich Supercomputing Centre, Institute for Complex Systems - Structural Biochemistry (ICS-6), Forschungszentrum Jülich GmbH, Jülich, Germany Search for more papers by this author Otto Berninghausen Otto Berninghausen Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Search for more papers by this author Holger Gohlke Corresponding Author Holger Gohlke [email protected] orcid.org/0000-0001-8613-1447 John von Neumann Institute for Computing, Jülich Supercomputing Centre, Institute for Complex Systems - Structural Biochemistry (ICS-6), Forschungszentrum Jülich GmbH, Jülich, Germany Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Roland Beckmann Corresponding Author Roland Beckmann [email protected] orcid.org/0000-0003-4291-3898 Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Search for more papers by this author Alexej Kedrov Corresponding Author Alexej Kedrov [email protected] orcid.org/0000-0001-9117-752X Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany Synthetic Membrane Systems, Institute for Biochemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Author Information Lukas Kater1, Benedikt Frieg2, Otto Berninghausen1, Holger Gohlke *,2,3, Roland Beckmann *,1 and Alexej Kedrov *,1,4 1Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany 2John von Neumann Institute for Computing, Jülich Supercomputing Centre, Institute for Complex Systems - Structural Biochemistry (ICS-6), Forschungszentrum Jülich GmbH, Jülich, Germany 3Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 4Synthetic Membrane Systems, Institute for Biochemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany *Corresponding author. Tel: +49 211 81 13662; E-mail: [email protected] *Corresponding author. Tel: +40 89 2180 76900; E-mail: [email protected] *Corresponding author. Tel: +49 211 81 13731; E-mail: [email protected] EMBO Reports (2019)20:e48191https://doi.org/10.15252/embr.201948191 See also: Y Tanaka & T Tsukazaki et al (October 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology. Synopsis Cryo-electron microscopy and atomistic simulations of SecYEG in the lipid environment reveal an early stage of membrane protein insertion. The flexible nascent chain triggers a conformational change that pre-opens the translocon. The complete structure of SecYEG translocon in the lipid bilayer is resolved. The bound ribosome:nascent chain opens the lateral gate of SecYEG at the cytoplasmic side. Nascent transmembrane domains remain flexible at the early insertion stage. SecY:SecE interactions may modulate the lateral gate dynamics. Introduction Membrane proteins constitute a large part of the cellular proteome and determine the vital functionality and identity of biological membranes. These proteins are co-translationally targeted as ribosome:nascent chain complexes (RNCs) to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in bacteria and archaea, where they are inserted by the dedicated and universally conserved Sec translocon (Fig 1A and B) 1. The translocon, an integral membrane protein itself, builds a protein-conducting channel in the lipid bilayer and allows either transmembrane passage of nascent polypeptide chains or their partitioning into the lipid environment as transmembrane α-helices (TMHs). The nascent chain hydrophobicity forms a basis for the triage 2. The central subunit of the translocon, SecY in bacteria or Sec61α in eukaryotes, consists of 10 TMHs arranged as a pseudo-symmetric "clam-shell" with a protein-conducting pore between the N- and C-terminal parts (Fig 1) 3, 4. A bilayer-facing crevice between SecY TMHs 2b and 7 is assumed to serve as a route, or a "lateral gate", for nascent TMHs to reach the hydrophobic membrane core. SecY is stabilized at the periphery by the essential subunit SecE/Sec61γ that contains two α-helices, one in interfacial and one in transmembrane topologies. SecE of some Gram-negative bacteria, including Escherichia coli, contains also an accessory pair of N-terminal TMHs, the role and localization of which have remained largely unclear 5. A non-essential and non-conserved SecG/Secβ subunit near the N-terminal half of SecY is built of either one or two TMHs and plays a stimulatory role in protein translocation 6. Figure 1. Structure and dynamics of SecYEG translocon Structure of quiescent SecYEG of Thermus thermophilus in the lipid cubic phase (PDB ID: 5AWW). TMHs 2b, 3, 7, and 8 of the lateral gate, as well as the proximate loop 6/7 involved in ribosome binding are indicated. The non-essential SecG subunit is omitted for clarity. Model of the SecY lateral gate opening upon inserting a nascent chain (red) in the lipid bilayer. The color-coding of SecYE TMHs is as in panel (A). In the presence of the completely inserted and folded nascent chain, TMHs 2b and 3 of the N-terminal domain of SecY are displaced (arrows) thus opening a broad passage for the nascent TMH toward the lipid moiety. SDS–PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated. Schematic drawing of a SecYEG-ND particle. Lateral dimensions of the nanodisc should be appropriate to accommodate a single SecYEG with surroundings lipids, thus mimicking the naturally occurring environment. Download figure Download PowerPoint The assembly of the translocon:ribosome complex at the cytoplasmic membrane interface is a key step in membrane protein biogenesis, as it allows the hydrophobic nascent chain to egress into the lipid bilayer via the translocon, while not being exposed to the polar aqueous environment 1, 7. The architecture of the complex has been extensively studied by structural methods, first of all cryo-electron microscopy (cryo-EM) 8-11. Binding of a ribosome results in minor rearrangements within the translocon and brings it to a pre-open or "primed" state 11. The following insertion of a sufficiently hydrophobic helical domain, such as a signal sequence or signal anchor domain, shifts the complete N-terminal domain of SecY/Sec61α by 22° and also tilts TMH 7, so the lateral gate of the translocon acquires an open state (Fig 1B) 12, 13. The folded signal sequence in a transbilayer topology may occupy the lateral gate where it replaces TMH 2b. Upon the further elongation of the nascent polypeptide chain, the newly inserted α-helix leaves the lateral gate and egresses into the lipid bilayer, and the translocon undergoes a reverse transition from a widely opened 14 to a compact, pre-closed state 15. Although the dynamics of the lateral gate have been commonly acknowledged 16, 17, the mechanism of the nascent chain insertion remains unclear. First, existing structures reflect rather late insertion stages, where the signal sequence has been fully inserted in the transmembrane topology, while early intermediates have been barely addressed 4, 18. Second, a vast majority of available ribosome:translocon structures represent detergent-solubilized complexes; however, the non-physiological environment and extensive downstream purification schemes may significantly affect the conformation and the interaction properties of membrane proteins, including the translocon 19-21. The variations in detergent-based solubilization protocols may explain contradictory results on the translocon dynamics, where either a local displacement of helices within the lateral gate or an extensive movement of the complete N-terminal half was observed upon the nascent chain insertion, and also the conformation of the central "plug" domain has been disputed 12, 13, 22. Furthermore, a compact "primed" state has been described for detergent-solubilized translocons in the absence of hydrophobic nascent chains 11, while a recent cryo-electron tomography analysis has revealed a predominantly open conformation of the ribosome-bound Sec61 within native ER membranes and so suggested a crucial effect of the molecular environment on protein dynamics 23. Up to date, the only structure of the translocon:ribosome complex at the lipid interface was obtained by cryo-EM when using nanodisc-reconstituted SecYEG (SecYEG-ND) bound to a translation-stalled RNC 14. Although demonstrating an advance compared to detergent-solubilized systems, the structure offers only limited resolution and also illustrates a rather late stage of the TMH insertion, with the translocon lateral gate widely open and the inserted anchor domain de-localized within the membrane. Here, we set out to determine the structure of the SecYEG:RNC complex that would describe an early stage of a transmembrane domain insertion into the lipid bilayer. Using cryo-EM and single-particle analysis, we resolved for the first time all three subunits of SecYEG in nanodiscs and described a novel conformation, where SecY TMHs 2b and 7 were apart at the cytoplasmic side to form a V-shaped lateral gate that is pre-opened for the nascent chain insertion, while accessory SecE TMHs 1 and 2 interacted with the gate at the periplasmic side. The RNC-induced dynamics within the translocon was validated by atomistic molecular dynamics simulations, which also described the interactions of SecYEG with anionic lipids. Cryo-EM data and site-specific chemical cross-linking further suggested that the FtsQ anchor domain is inserted via the lateral gate, where it forms close contacts with SecY TMH 7, but remains highly flexible before leaving the translocon. Results and Discussion Functional reconstitution of E. coli SecYEG in nanodiscs has been previously performed by several groups for biochemical, biophysical, and structural studies and allowed probing of the translocon interactions with the motor protein SecA, targeting factors, and ribosomes 14, 20, 24, 25. The diameter of formed nanodiscs is essentially determined by the length of the major scaffold protein (MSP) that girdles the lipid bilayer 26, 27. Translocon molecules have been initially embedded into nanodiscs as small as 9 nm in diameter 16, 20, 24. However, a follow-up functional analysis demonstrated that larger nanodisc dimensions are beneficial for facilitating the translocation activity, likely due to the increased amount of co-reconstituted lipids 25, 28. Thus, we used an extended scaffold protein MSP1E3D1 and POPG/POPC lipids to reconstitute SecYEG into nanodiscs with a diameter of approximately 12 nm. A large excess of MSPs and lipids ensured that translocons were reconstituted predominantly as monomers 25, as those have been shown to be the principle functional form both in bacteria and in eukaryotes 9, 29, 30. Due to solvent-exposed loops of SecYEG, which contributed to the hydrodynamic radius, SecYEG-ND could be separated from "empty" nanodiscs containing only lipids by means of size-exclusion chromatography (Fig 1C). Within formed nanodiscs, SecYEG would occupy ~30% of the surface area (Fig 1D) 25, 26, 28, thus providing sufficient space for the conformational dynamics, and for insertion of nascent TMHs upon interactions with RNCs. We have previously demonstrated that SecYEG:ribosome assembly is strongly enhanced by hydrophobic nascent chains, such as a TMH of FtsQ, a model protein for studying the SecYEG-mediated insertion pathway 20. The hydrophobic polypeptide exposed from a ribosome exit tunnel is sufficient to mediate SecYEG:ribosome binding in native and model membranes, even in the absence of targeting factors 20, 31, but unlikely to undergo the complete insertion due to its short ribosome-bound linker. Thus, to investigate an early stage of the TMH insertion, we prepared translation-stalled ribosomes, which exposed the first 48 amino acids of FtsQ, including the TMH within the nascent chain (Fig EV1), and incubated those with a 10-fold excess of SecYEG-ND to achieve complex formation. After vitrification, samples were subjected to cryo-EM imaging and single-particle analysis. RNCs could be readily seen in raw micrographs, and a discoidal density of SecYEG-ND bound to RNCs was observed in projection groups of two-dimensional (2D) classification and in 3D reconstructions (Fig 2A–C). After sorting and refinement steps (Fig EV2), the ribosome structure was resolved at 3.3 Å, and independent refinement of the SecYEG-ND:RNC complex elements led to 3.2 and 3.1 Å resolution for the small (30S) and large (50S) ribosomal subunits, respectively (Appendix Fig S1), and was limited to 6 Å for the lipid-embedded SecYEG due to its small size and apparent dynamics relative to the 50S ribosomal subunit (Movie EV1). The local resolution within the SecYEG-ND particle ranged from 3.5 Å at the ribosome contact sites to 6–7 Å within the transmembrane core and above 10 Å for the surrounding MSP1E3D1 and lipid head groups, which could be visualized at lower threshold levels (Fig 2D and E). Click here to expand this figure. Figure EV1. Preparation of RNC FtsQ The architecture of FtsQ nascent chain: N-terminal hexa-histidine tag was fused via 3C protease cleavage site to the N-terminal domain of FtsQ followed by hemagglutinin (HA) recognition tag and TnaC stalling sequence. The length (amino acids) of each segment is indicated. Coomassie-stained protein content of isolated RNC FtsQ (left) and Western blot against the HA tag within the nascent chain (right). Western blot visualizes a complex of the FtsQ nascent chain (109 aa, ˜12 kDa) and tRNA (˜25 kDa). Putative architecture of RNC:SecYEG-ND complex upon the insertion of the early intermediate of FtsQ. Download figure Download PowerPoint Figure 2. Cryo-EM of the RNC FtsQ:SecYEG-ND complex Representative cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled. Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles. Three-dimensional reconstruction of RNC FtsQ:SecYEG-ND complex. Primary structural elements of the ribosome and SecYEG-ND are indicated. Local resolution map of SecYEG-ND sub-particle. The cytoplasmic side of the translocon demonstrates higher resolution due to stabilization by the bound ribosome, while high resolution at the periplasmic side is hindered by the SecYEG-ND dynamics within the complex. The associated ribosome is not shown for clarity. A planar slice through the SecYEG-ND core at different signal levels (blue/green/red) with indicated positions of SecYEG TMHs (SecY indicated in orange, SecE in purple, and SecG in green). A single helical turn could be fitted in a density in the area where SecG TMH 1 was expected (green asterisk). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Sorting and refinement scheme of RNC FtsQ:SecYEG-ND complexesParticle picking was performed with gautomatch, all downstream processing was performed in Relion 3.0 beta. Dataset 1 and Dataset 2 were joined after initial 3D classification, where ribosomal particles with P-site tRNA and SecYEG-ND were selected from both datasets. Initial processing steps were performed with 2× binned data for stronger signal-to-noise ratio at SecYEG-ND. Download figure Download PowerPoint In agreement with the initial prediction, the nanodisc dimensions were sufficiently large to accommodate a single copy of SecYEG. As SecYEG was positioned in the center of the nanodisc and contacts with edges of the lipid bilayer or MSP were not observed, it is likely that the translocon conformation was not affected by the confined environment. As electron densities of the centrally positioned translocon and the MSP were well-separated (Fig 2E), it facilitated the assignment of rod-shaped densities to TMHs of SecYEG and building the molecular model based on the structure of the quiescent translocon 4. Both TMHs and extramembrane domains of SecY, SecE, and SecG subunits could be unambiguously fitted into the cryo-EM density (Figs 2E and 3A). The translocon:ribosome complex was established via the well-known canonical interactions 9, 11, 14: Two structured cytoplasmic loops between TMHs 6/7 and 8/9 of SecY extended toward the ribosomal tunnel to interact with rRNA helices H6, H24, and H50, and the uL23 protein. Additionally, the ribosomal protein uL24 approached the C-terminal end of the SecY TMH 10, and the ribosomal protein uL23 formed two contacts within the essential amphipathic helix of SecE. Differently to earlier findings 14, we did not observe the contact between the rRNA helix H59 and the lipid head groups, although the H59 helix was displaced toward the bilayer (Fig 3B). It seems plausible that those contacts are established at a later stage of membrane protein insertion, when one or more nascent TMHs egress the lipid bilayer and the H59 helix "screens" the charge of connecting loops, and so participates in the topology determination 15, 32. When evaluating other known structures of bacterial and eukaryotic translocons in complex with ribosomes (Appendix Fig S2), we noted a close agreement between our model and the detergent-solubilized E. coli SecYEG bound to a translation-stalled ribosome 18. Interestingly, although the SecYEG structures in both environments were highly similar, the relative orientation of the ribosome and SecYEG differed substantially: While being bound to the RNC via its C-terminal domain, the detergent-solubilized translocon rotated as a rigid body away from the rRNA helix H59, so the displacement was most pronounced for its N-terminal half (Fig EV3). It is tempting to speculate that the altered SecYEG:ribosome binding geometry, as well as the enhanced affinity of the complex in detergent 20, arose from the lack of electrostatic interactions between the rRNA and the polar moiety of lipid head groups. Figure 3. Structural dynamics of the translocon and ribosome upon the nascent chain insertion Isolated cryo-EM density of SecYEG with the fitted molecular model of the translocon in front and the cytoplasmic views. SecY is displayed in rainbow pattern, SecE in purple, and SecG in green. Dashed box: a contact site between tilted SecE TMHs 1/2 and SecY TMH 8. Cryo-EM density corresponding to the ribosomal RNA helix 59 ("H59") is displaced toward the nanodisc. No contact with the lipid bilayer could be detected. Blue ribbon: structure of the translocon-free 50S ribosomal subunit (PDB ID: 4UY8). Central cross-section through the SecYEG model. The "plug" TMH 2a occupies the central position, thus keeping the translocon sealed. The nanodisc perimeter is indicated as a dashed circle. The lateral gate of nanodisc-embedded translocon undergoes rearrangements relatively to a quiescent conformation (left, PDB ID 5AWW) and an RNC-bound detergent-solubilized state (right, PDB ID 5GAE). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Environment determines the architecture of RNC: translocon complex Superimposition of SecYEG-ND (colored) and detergent-solubilized translocon (gray, PDB ID: 5GAE) reveals different alignments if either 50S ribosome subunit (left) or the translocon itself (right) was used as an alignment template. Depending on the environment, the translocon undergoes a rotation around the ribosome-bound loop 6/7. The movement directions are indicated by rods, and the magnitude, as measured for positions of Cα atoms, by color (central panel). Download figure Download PowerPoint In spite of the loose binding of SecYEG to the RNC and its higher flexibility, the complete architecture of essential SecY and SecE subunits was resolved, and a single-helix density proximate to the SecY N-terminal domain was assigned to TMH 2 of the SecG subunit, while TMH 1 could not be reliably detected (Figs 2E and 3A). No SecG subunit could be resolved in the earlier structure of SecYEG-ND 14, and the crystal structure of the quiescent SecYEG revealed that SecG TMH 1 faces away from the translocon core, so its periplasmic tip is separated by ~10 Å from the nearest TMH 4 of SecY, with a lipid molecule filling the void 4. Thus, weak protein:protein inter-subunit interactions in the lipid environment likely favor spatial dynamics of SecG, up to a complete topology inversion 33, and the dynamics might be modulated by the ribosome binding. Remarkably, within the SecYEG-ND complex we could clearly observe accessory TMHs 1 and 2 of SecE, which were either absent or only poorly resolved in previous translocon structures 14, 15, 18. Earlier models placed the SecE TMHs either distanced from the translocon by 20 Å, or near SecY TMH 9, i.e., at the back of the translocon 14, 15 (Appendix Fig S2). However, our structure revealed a very different organization of the complex, as SecE TMHs formed a helical hairpin in close proximity to SecY C-terminal domain, and the hairpin was tilted within the lipid bilayer by ~30° (Fig 3A). Such a tilted orientation of the SecE TMHs could also be recognized in densely packed 2D crystals of SecYEG 34, 35, but has not been reported for either free-standing or ribosome-bound translocons. Surprisingly, the periplasmic loop of the SecE helical hairpin reached TMH 8 and a short helix connecting TMHs 7 and 8 of SecY, and so appeared in direct contact with the lateral gate of the translocon, thus suggesting a potential role of SecE in the translocon gating mechanism but also explaining interactions of SecE with nascent TMHs soon after their membrane partitioning 36. We further examined whether the early interactions with the RNC were sufficient to trigger a conformational change within SecYEG, as it would be required for the nascent chain insertion into the lipid bilayer. SecY TMH 2a, known as a plug domain 37, 38, resided in the central position, thus keeping the SecY pore sealed upon RNC binding 12, 31, and only minor shifts could be seen for most TMHs in comparison with the quiescent state or detergent-solubilized SecYEG:RNC complex 4, 18 (Fig 3C and Appendix Fig S2). Interestingly though, substantial rearrangements were observed within the lateral gate of the translocon, when compared both to the quiescent and to RNC-bound detergent-solubilized states (Fig 3D): TMH 2b was displaced toward the central pore of the translocon, and SecY TMH 7 underwent a tilting of ~ 5°, so its cytoplasmic and the periplasmic ends approached TMH 8 and TMH 3, respectively 3, 4. This tilting of TMH 7 was coupled to a displacement of TMH 8, as they are connected via a short rigid helix at the periplasmic side (Fig 3D). The resulting conformation of the ribosome-bound translocon manifested a V-shaped crevice at the cytoplasmic side of the lateral gate that differed from the rather closed conformation of the detergent-solubilized SecYEG 18, but also from "primed" and fully opened post-insertion states of the eukaryotic homolog 10, 11, 13. Thus, the observed conformation likely reflected a novel early stage in the gate opening. Such dynamics are in agreement with a previous fluorescence-based study on SecYEG-ND:RNC 16, but, to our knowledge, represent the first direct visualization of the pre-opened translocon in the lipid environment. To investigate whether the observed translocon conformation was a result of RNC FtsQ binding, we employed microsecond-long molecular dynamics (MD) simulations of SecYEG in explicit solvent and an explicit membrane, which allows to study the behavior of lipid-embedded SecYEG in full atomic detail 39. From the projection of MD conformations of SecY onto the plane spanned by the first two principal components (PC; both PCs together describe ~50% of the total variance of motions during the simulations), a configurational free energy landscape was computed (equation 1). In this landscape, the SecY conformation from the SecYEG-ND:RNC complex lies in an area of slightly elevated free energy (ΔGconf.,i ≈ 2 kcal/mol, Appendix Fig S3A), suggesting that this conformation was stabilized by the bound RNC and/or the nascent chain. The mechanism of structural adaptation of the translocon was then probed in a reverse direction, as the MD simulations started from the RNC-bound SecYEG conformation, but without RNC FtsQ. That way, the adaptation toward a non-disturbed quiescent state could be followed, as has previously been shown for membrane protein complexes 40, 41. The cytoplasmic loop 6/7 of SecY was highly mobile (mean root-mean-square fluctuations (RMSF) > 5 Å; Fig 4A), likely due to the absent ribosome that otherwise recruits the loop as a docking site. The TMHs were substantially less dynamic (RMSF < 3 Å), except for the lateral gate and the cytoplasmic part of TMH 2b. Structural differences upon reaching the free energy minimum were the most substantial for loop 6/7 and were followed by the lateral gate (Fig 4B). We measured internal distances within the lateral gate (TMHs 2, 7, and 8), between TMH 7 and the adjacent TMH 3, as well as the angle η between TMH 7 and TMH 8 (Fig EV4, panels A and C). The cryo-EM structure implied that binding of RNC FtsQ to SecY induced tilting of TMH 7, such that its periplasmic end approached TMH 3, while TMH 2b shifted toward the pore. This effect was completely reversed in the absence of the RNC, as both the distance between TMHs 3 and 7 and the angle η increased (Fig EV4, panels B and D). Compared to the initial conformation, the distances between TMHs 2b and 7, and between TMHs 2 and 8, decreased over the course of the simulations, while the distance between TMHs 7 and 8 increased, which led to a closing of the observed V-shaped crevice (Fig EV4, panel B). Interestingly, the PC analysis also suggested that the movements of TMHs 7 and 8 were connected to the dynamics of the cytoplasmic loop 6/7 (Fig 4C, Appendix Fig S3B), such that the ribosome binding likely also influences the structural dynamics within the lateral gate, in agreement with an earlier structure of the ribosome-bound Sec61 translocon 11 and the recent biochemical data 42. In the absence of a ribosome, binding of a short signal peptide causes an outward displacement of TMH 2b but not TMH 7 4, so the enhanced structural dynamics at the cytoplasmic side of the lateral gate likely allows a range of pre-opened translocon conformations. Figure 4. Analysis of molecul
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