RESUMO
The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Canais de Translocação SEC/química , Proteínas SecA/metabolismo , Proteínas de Bactérias/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Transporte Proteico , Nucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
Secretory proteins are cotranslationally or posttranslationally translocated across lipid membranes via a protein-conducting channel named SecY in prokaryotes and Sec61 in eukaryotes. The vast majority of secretory proteins in bacteria are driven through the channel posttranslationally by SecA, a highly conserved ATPase. How a polypeptide chain is moved by SecA through the SecY channel is poorly understood. Here, we report electron cryomicroscopy structures of the active SecA-SecY translocon with a polypeptide substrate. The substrate is captured in different translocation states when clamped by SecA with different nucleotides. Upon binding of an ATP analog, SecA undergoes global conformational changes to push the polypeptide substrate toward the channel in a way similar to how the RecA-like helicases translocate their nucleic acid substrates. The movements of the polypeptide substrates in the SecA-SecY translocon share a similar structural basis to those in the ribosome-SecY complex during cotranslational translocation.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Proteínas SecA/metabolismo , Proteínas de Bactérias/metabolismo , Canais de Translocação SEC/metabolismo , Modelos Moleculares , Transporte Proteico , Peptídeos/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly "catch and release" trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted.
Assuntos
Adenosina Trifosfatases/metabolismo , Transporte Biológico/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas SecA/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas/fisiologia , Canais de Translocação SEC/metabolismoRESUMO
The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bilayers. The association of SecA with SecYEG, forming membrane-bound SecYEG/SecA translocases, is directly visualized. Recent work showing topographical aspects of the translocation process that vary with precursor species is also discussed. The data suggests that the Sec system does not employ a single translocation mechanism. We posit that differences in the spatial frequency distribution of hydrophobic content within precursor sequences may be a determining factor in mechanism selection. Precise AFM investigations of active translocases are poised to advance our currently vague understanding of the complicated macromolecular movements underlying protein export across membranes.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas SecA/metabolismo , Microscopia de Força Atômica , Canais de Translocação SEC/metabolismo , Transporte Proteico , Proteínas de Membrana/metabolismoRESUMO
Most bacterial secretory proteins destined to the extracytoplasmic space are secreted posttranslationally by the Sec translocase. SecA, a key component of the Sec system, is the ATPase motor protein, directly responsible for transferring the preprotein across the cytoplasmic membrane. SecA is a large protein, composed of several domains, capable of binding client preproteins and a variety of partners, including the SecYEG inner membrane channel complex, membrane phospholipids and ribosomes. SecA-mediated translocation can be divided into two major steps: (1) targeting of the preproteins to the membrane translocation apparatus and (2) transport across the membrane through the SecYEG channel. In this review we present current knowledge regarding SecA structure and function of this protein in both translocation steps. The most recent model of the SecA-dependent preprotein mechanical translocation across the bacterial cytoplasmic membrane is described. A possibility of targeting SecA with inhibitory compounds as a strategy to combat pathogenic bacteria will be discussed as well.
Assuntos
Proteínas de Escherichia coli/metabolismo , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Adenosina Trifosfatases/metabolismo , Antibacterianos/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte ProteicoRESUMO
The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity-a prerequisite for understanding how biological systems work-has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (â¼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.
Assuntos
Trifosfato de Adenosina/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Biológicos , Proteínas SecA/metabolismo , Bactérias/citologia , Bioensaio/métodos , Hidrólise , Cinética , Bicamadas Lipídicas/metabolismo , Luciferases/químicaRESUMO
BACKGROUND: The SecA DEAD (Asp-Glu-Ala-Asp) motor protein uses binding and hydrolysis of adenosine triphosphate (ATP) to push secretory proteins across the plasma membrane of bacteria. The reaction coordinate of nucleotide exchange is unclear at the atomic level of detail. METHODS: We performed multiple atomistic computations of the DEAD motor domain of SecA with different occupancies of the nucleotide and magnesium ion sites, for a total of ~1.7 µs simulation time. To characterize dynamics at the active site we analyzed hydrogen-bond networks. RESULTS: ATP and ADP can bind spontaneously at the interface between the nucleotide binding domains, albeit at an intermediate binding site distinct from the native site. Binding of the nucleotide is facilitated by the presence of a magnesium ion close to the glutamic group of the conserved DEAD motif. In the absence of the magnesium ion, protein interactions of the ADP molecule are perturbed. CONCLUSIONS: A protein hydrogen-bond network whose dynamics couples to the occupancy of the magnesium ion site helps guide the nucleotide along the nucleotide exchange path. In SecA, release of magnesium might be required to destabilize the ADP binding site prior to release of the nucleotide. GENERAL SIGNIFICANCE: We identified dynamic hydrogen-bond networks that help control nucleotide exchange in SecA, and stabilize ADP at an intermediate site that could explain slow release. The reaction coordinate of the protein motor involves complex rearrangements of a hydrogen-bond network at the active site, with perturbation of the magnesium ion site likely occurring prior to the release of ADP.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas SecA/metabolismo , Sítios de Ligação , Cátions Bivalentes/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Ligação de Hidrogênio , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas SecA/químicaRESUMO
SecA is an essential component of the Sec protein secretion pathway in bacteria. Secretory proteins targeted to the Sec pathway by their N-terminal signal peptide bind to SecA, which couples binding and hydrolysis of adenosine triphosphate with movement of the secretory protein across the membrane-embedded SecYEG protein translocon. The phylogenetic diversity of bacteria raises the important question as to whether the region of SecA where the pre-protein binds has conserved sequence features that might impact the reaction mechanism of SecA. To address this question we established a large data set of SecA protein sequences and implemented a protocol to cluster and analyze these sequences according to features of two of the SecA functional domains, the protein binding domain and the nucleotide-binding domain 1. We identify remarkable sequence diversity of the protein binding domain, but also conserved motifs with potential role in protein binding. The N-terminus of SecA has sequence motifs that could help anchor SecA to the membrane. The overall sequence length and net estimated charge of SecA sequences depend on the organism.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas SecA/metabolismo , Análise por Conglomerados , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/classificação , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/classificação , Filogenia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas SecA/química , Proteínas SecA/classificação , Análise de Sequência de ProteínaRESUMO
The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.
Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Proteínas SecA/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas SecA/genética , Azida Sódica/farmacologiaRESUMO
Many nascent polypeptides synthesized in the cytoplasm are translocated across membranes via a specific 'translocon' composed of protein complexes. Recently, a novel targeting pathway for the outer membrane ß-barrel proteins (OMPs) in Gram-negative bacteria was discovered. The cell envelope of Gram-negative bacteria is composed of the inner (plasma) membrane (IM) and the outer membrane (OM). In this new pathway, a SecAN protein, which is mainly present in the IM as a homo-oligomer, translocates nascent OMPs across the IM; at the same time, SecAN directly interacts with the ß-barrel assembly machinery (BAM) complex embedded within the OM. A supercomplex (containing SecAN , the BAM complex and many other proteins) spans the IM and OM, and is involved in the biogenesis of OMPs. Investigation of the function of SecAN and the supercomplex, as well as the translocation mechanism, will require elucidation of their structures. However, no such structures are available. Therefore, here, I describe the use of protein modeling to build homology models for SecAN and theoretical structures for the core-complex composed of SecAN and the BAM complex, which is a key part of the supercomplex. The modeling data are consistent with previous experimental observations and demonstrated a conformational change of the core-complex. I conclude by proposing mechanisms for how SecAN and the supercomplex function in the biogenesis of OMPs.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Bactérias Gram-Negativas/enzimologia , Modelos Químicos , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Proteínas SecA/química , Proteínas SecA/metabolismo , Algoritmos , Motivos de Aminoácidos/genética , Citoplasma/metabolismo , Mutação , Conformação Proteica em Folha beta , Multimerização Proteica , Transporte Proteico , Proteínas SecA/genética , Transdução de SinaisRESUMO
Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.
Assuntos
Canais de Translocação SEC/metabolismo , Proteínas SecA/química , Proteínas SecA/metabolismo , Adenosina Trifosfatases/química , Ânions/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Fosfolipídeos/química , Sinais Direcionadores de Proteínas , Transporte Proteico , Canais de Translocação SEC/química , Proteínas SecA/fisiologiaRESUMO
The bacterial Sec translocon is a multi-protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Espectrometria de Massa com Troca Hidrogênio-Deutério , Nucleotídeos/metabolismo , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/química , Ativação do Canal Iônico , Conformação Proteica , Canais de Translocação SEC/química , Proteínas SecA/químicaRESUMO
Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Bicamadas Lipídicas/química , Proteínas de Transporte de Monossacarídeos/química , Proteínas Periplásmicas de Ligação/química , Precursores de Proteínas/química , Proteínas SecA/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Multimerização Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Transporte Proteico , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC/química , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Proteínas SecA/genética , Proteínas SecA/metabolismoRESUMO
In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Translocação Bacteriana , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas SecA/química , Proteínas SecA/metabolismo , Sequência de Aminoácidos , Biocatálise , Reagentes de Ligações Cruzadas/química , Evolução Molecular , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/metabolismo , Filogenia , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Ribossomos/metabolismo , Especificidade por SubstratoRESUMO
DExD/H-box proteins are soluble enzymes that couple binding and hydrolysis of adenosine triphosphate (ATP) with reactions involving RNA metabolism or bind and push newly synthesized proteins across bacterial cell membranes. Knowledge of the reaction mechanism of these enzymes could help the development of new therapeutics. In order to explore the mechanism of long-distance conformational coupling in SecA, the DEAD-box motor of the Sec protein secretion in bacteria, we implemented algorithms that provide simplified graph representations of the protein's dynamic hydrogen-bond networks. We find that mutations near the nucleotide-binding site or changes of the nucleotide-binding state of SecA associate with altered dynamics at the preprotein binding domain and identify extended networks of hydrogen bonds that connect the active site of SecA to the region where SecA binds newly synthesized secretory proteins. Water molecules participate in hydrogen-bonded water chains that bridge functional domains of SecA and could contribute to long-distance conformational coupling.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas SecA/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/química , Proteínas de Bactérias/química , Sítios de Ligação , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas SecA/química , Água/química , Água/metabolismoRESUMO
More than a third of all bacterial polypeptides, comprising the 'exportome', are transported to extracytoplasmic locations. Most of the exportome is targeted and inserts into ('membranome') or crosses ('secretome') the plasma membrane. The membranome and secretome use distinct targeting signals and factors, and driving forces, but both use the ubiquitous and essential Sec translocase and its SecYEG protein-conducting channel. Membranome export is co-translational and uses highly hydrophobic N-terminal signal anchor sequences recognized by the signal recognition particle on the ribosome, that also targets C-tail anchor sequences. Translating ribosomes drive movement of these polypeptides through the lateral gate of SecY into the inner membrane. On the other hand, secretome export is post-translational and carries two types of targeting signals: cleavable N-terminal signal peptides and multiple short hydrophobic targeting signals in their mature domains. Secretome proteins remain translocation competent due to occupying loosely folded to completely non-folded states during targeting. This is accomplished mainly by the intrinsic properties of mature domains and assisted by signal peptides and/or chaperones. Secretome proteins bind to the dimeric SecA subunit of the translocase. SecA converts from a dimeric preprotein receptor to a monomeric ATPase motor and drives vectorial crossing of chains through SecY aided by the proton motive force. Signal peptides are removed by signal peptidases and translocated chains fold or follow subsequent trafficking.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Chaperonas Moleculares , Sinais Direcionadores de Proteínas , Canais de Translocação SEC , Proteínas SecA , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Transporte Proteico , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Proteínas SecA/química , Proteínas SecA/metabolismoRESUMO
SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptídeos/metabolismo , Proteínas SecA/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Proteínas SecA/químicaRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Dobramento de Proteína , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Precursores de Proteínas/metabolismo , Transporte Proteico , Canais de Translocação SEC/química , Proteínas SecA/químicaRESUMO
SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.