Structure of the posttranslational Sec protein-translocation channel complex from yeast.
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ABSTRACT:
SUBMITTER: Itskanov S
PROVIDER: S-EPMC6760253 | biostudies-literature | 2019 Jan
REPOSITORIES: biostudies-literature
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Project description:Proteins synthesized in the cytosol either remain there or are localized to a specific membrane and subsequently translocated to another cellular compartment. These extracytosolic proteins have to cross, or be inserted into, a phospholipid bilayer-a process governed by membrane-bound protein transporters designed to recognize and receive appropriate polypeptides and thread them through the membrane. One such translocation complex, SecY/Sec61, is found in every cell, in either the plasma membrane of bacteria and archaea or the endoplasmic reticulum membrane of eukaryotes. Recent structural findings, combined with previous genetic and biochemical studies, have helped to describe how the passage of proteins through the membrane might occur, but several points of uncertainty remain.
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Project description:Most proteins are secreted from bacteria by the interaction of the cytoplasmic SecA ATPase with a membrane channel, formed by the heterotrimeric SecY complex. Here we report the crystal structure of SecA bound to the SecY complex, with a maximum resolution of 4.5 ångström (A), obtained for components from Thermotoga maritima. One copy of SecA in an intermediate state of ATP hydrolysis is bound to one molecule of the SecY complex. Both partners undergo important conformational changes on interaction. The polypeptide-cross-linking domain of SecA makes a large conformational change that could capture the translocation substrate in a 'clamp'. Polypeptide movement through the SecY channel could be achieved by the motion of a 'two-helix finger' of SecA inside the cytoplasmic funnel of SecY, and by the coordinated tightening and widening of SecA's clamp above the SecY pore. SecA binding generates a 'window' at the lateral gate of the SecY channel and it displaces the plug domain, preparing the channel for signal sequence binding and channel opening.
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Project description:Direct simulations reveal key mechanistic features of early-stage protein translocation and membrane integration via the Sec-translocon channel. We present a novel computational protocol that combines non-equilibrium growth of the nascent protein with microsecond timescale molecular dynamics trajectories. Analysis of multiple, long timescale simulations elucidates molecular features of protein insertion into the translocon, including signal-peptide docking at the translocon lateral gate (LG), large lengthscale conformational rearrangement of the translocon LG helices, and partial membrane integration of hydrophobic nascent-protein sequences. Furthermore, the simulations demonstrate the role of specific molecular interactions in the regulation of protein secretion, membrane integration, and integral membrane protein topology. Salt-bridge contacts between the nascent-protein N-terminus, cytosolic translocon residues, and phospholipid head groups are shown to favor conformations of the nascent protein upon early-stage insertion that are consistent with the Type II (N(cyt)/C(exo)) integral membrane protein topology, and extended hydrophobic contacts between the nascent protein and the membrane lipid bilayer are shown to stabilize configurations that are consistent with the Type III (N(exo)/C(cyt)) topology. These results provide a detailed, mechanistic basis for understanding experimentally observed correlations between integral membrane protein topology, translocon mutagenesis, and nascent-protein sequence.
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Project description:Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome-nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryo-electron microscopy. Non-translating ribosome-SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.
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Project description:Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.
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Project description:We present a coarse-grained modeling approach that spans the nanosecond- to minute-timescale dynamics of cotranslational protein translocation. The method enables direct simulation of both integral membrane protein topogenesis and transmembrane domain (TM) stop-transfer efficiency. Simulations reveal multiple kinetic pathways for protein integration, including a mechanism in which the nascent protein undergoes slow-timescale reorientation, or flipping, in the confined environment of the translocon channel. Competition among these pathways gives rise to the experimentally observed dependence of protein topology on ribosomal translation rate and protein length. We further demonstrate that sigmoidal dependence of stop-transfer efficiency on TM hydrophobicity arises from local equilibration of the TM across the translocon lateral gate, and it is predicted that slowing ribosomal translation yields decreased stop-transfer efficiency in long proteins. This work reveals the balance between equilibrium and nonequilibrium processes in protein targeting, and it provides insight into the molecular regulation of the Sec translocon.
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Project description:Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.
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Project description:An important aspect of cellular function is the correct targeting and delivery of newly synthesized proteins. Central to this task is the machinery of the Sec translocon, a transmembrane channel that is involved in both the translocation of nascent proteins across cell membranes and the integration of proteins into the membrane. Considerable experimental and computational effort has focused on the Sec translocon and its role in nascent protein biosynthesis, including the correct folding and expression of integral membrane proteins. However, the use of molecular simulation methods to explore Sec-facilitated protein biosynthesis is hindered by the large system sizes and long (i.e., minute) time scales involved. In this work, we describe the development and application of a coarse-grained simulation approach that addresses these challenges and allows for direct comparison with both in vivo and in vitro experiments. The method reproduces a wide range of experimental observations, providing new insights into the underlying molecular mechanisms, predictions for new experiments, and a strategy for the rational enhancement of membrane protein expression levels.
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Project description:Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.
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