Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon.

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.

Structural basis of SecA-mediated protein translocation.

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.

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism.

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.

Atomic Force Microscopy Reveals Complexity Underlying General Secretory System Activity.

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.

SecA - a multidomain and multitask bacterial export protein.

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.

Discovery of novel SecA inhibitors against "Candidatus Liberibacter asiaticus" through virtual screening and biological evaluation.

"Candidatus Liberibacter asiaticus" (Ca. L. asiaticus) is the causal agent of Huanglongbing disease of citrus and current study focuses on the discovery of novel small-molecule inhibitors against SecA protein of Ca. L. asiaticus. In this study, homologous modeling was used to construct the three-dimensional structure of SecA. Then, molecular docking-based virtual screening and two rounds of in vitro bacteriostatic experiments were utilized to identify novel small-molecule inhibitors of SecA. Encouragingly, 93 compounds were obtained and two of them (P684-2850, P684-3808) showed strong antimicrobial activities against Liberibacter crescens BT-1 in bacteriostatic experiments. Finally, molecular dynamics simulations were employed to explore the binding modes of the receptor-ligand complexes. Results in MD simulations showed that compound P684-3808 was relatively stable during simulation, while compound P684-2850 left the binding pocket. Compound P684-3808 might be suitable as a lead compound for further development of antimicrobial compounds against SecA of Ca. L. asiaticus.

A nexus of intrinsic dynamics underlies translocase priming.

The cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We show that priming exploits a nexus of intrinsic dynamics in SecA. Using atomistic simulations, smFRET, and HDX-MS, we reveal multiple dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are functionally important and conserved. Central to the nexus is a slender stem through which rotation of the preprotein clamp of SecA is biased by ATPase domain motions between open and closed clamping states. An H-bonded framework covering most of SecA enables multi-tier dynamics and conformational alterations with minimal energy input. As a result, cognate ligands select preexisting conformations and alter local dynamics to regulate catalytic activity and clamp motions. These events prime the translocase for high-affinity reception of non-folded preprotein clients. Dynamics nexuses are likely universal and essential in multi-liganded proteins.

Cellular dynamics of the SecA ATPase at the single molecule level.

In bacteria, the SecA ATPase provides the driving force for protein secretion via the SecYEG translocon. While the dynamic interplay between SecA and SecYEG in translocation is widely appreciated, it is not clear how SecA associates with the translocon in the crowded cellular environment. We use super-resolution microscopy to directly visualize the dynamics of SecA in Escherichia coli at the single-molecule level. We find that SecA is predominantly associated with and evenly distributed along the cytoplasmic membrane as a homodimer, with only a minor cytosolic fraction. SecA moves along the cell membrane as three distinct but interconvertible diffusional populations: (1) A state loosely associated with the membrane, (2) an integral membrane form, and (3) a temporarily immobile form. Disruption of the proton-motive-force, which is essential for protein secretion, re-localizes a significant portion of SecA to the cytoplasm and results in the transient location of SecA at specific locations at the membrane. The data support a model in which SecA diffuses along the membrane surface to gain access to the SecYEG translocon.

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover.

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 (∼30 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.

The SecA motor generates mechanical force during protein translocation.

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.

Molecular movie of nucleotide binding to a motor protein.

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.

The SecA ATPase motor protein binds to Escherichia coli liposomes only as monomers.

The essential SecA motor ATPase acts in concert with the SecYEG translocon to secrete proteins into the periplasmic space of Escherichia coli. In aqueous solutions, SecA exists largely as dimers, but the oligomeric state on membranes is less certain. Crystallographic studies have suggested several possible solution dimeric states, but its oligomeric state when bound to membranes directly or indirectly via the translocon is controversial. We have shown using disulfide crosslinking that the principal solution dimer, corresponding to a crystallographic dimer (PDB 1M6N), binds only weakly to large unilamellar vesicles (LUV) formed from E. coli lipids. We report here that other soluble crosslinked crystallographic dimers also bind weakly, if at all, to LUV. Furthermore, using a simple glutaraldehyde crosslinking scheme, we show that SecA is always monomeric when bound to LUV formed from E. coli lipids.

Cardiolipin is required in vivo for the stability of bacterial translocon and optimal membrane protein translocation and insertion.

Translocation of preproteins across the Escherichia coli inner membrane requires anionic lipids by virtue of their negative head-group charge either in vivo or in situ. However, available results do not differentiate between the roles of monoanionic phosphatidylglycerol and dianionic cardiolipin (CL) in this essential membrane-related process. To define in vivo the molecular steps affected by the absence of CL in protein translocation and insertion, we analyzed translocon activity, SecYEG stability and its interaction with SecA in an E. coli mutant devoid of CL. Although no growth defects were observed, co- and post-translational translocation of α-helical proteins across inner membrane and the assembly of outer membrane ß-barrel precursors were severely compromised in CL-lacking cells. Components of proton-motive force which could impair protein insertion into and translocation across the inner membrane, were unaffected. However, stability of the dimeric SecYEG complex and oligomerization properties of SecA were strongly compromised while the levels of individual SecYEG translocon components, SecA and insertase YidC were largely unaffected. These results demonstrate that CL is required in vivo for the stability of the bacterial translocon and its efficient function in co-translational insertion into and translocation across the inner membrane of E. coli.

Iron is a ligand of SecA-like metal-binding domains in vivo.

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.

Diversity and sequence motifs of the bacterial SecA protein motor.

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.

Structural insights into the mechanism of a novel protein targeting pathway in Gram-negative bacteria.

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.

Binding of SecA ATPase monomers and dimers to lipid vesicles.

The Escherichia coli SecA ATPase motor protein is essential for secretion of proteins through the SecYEG translocon into the periplasmic space. Its function relies upon interactions with the surrounding lipid bilayer as well as SecYEG translocon. That negatively charged lipids are required for bilayer binding has been known for >25 years, but little systematic quantitative data is available. We have carried out an extensive investigation of SecA partitioning into large unilamellar vesicles (LUV) using a wide range of lipid and electrolyte compositions, including the principal cytoplasmic salt of E. coli, potassium glutamate, which we have shown stabilizes SecA. The water-to-bilayer transfer free energy is about -7.5 kcal mol-1 for typical E. coli lipid compositions. Although it has been established that SecA is dimeric in the cytoplasm, we find that the most widely cited dimer form (PDB 1M6N) binds only weakly to LUVs formed from E. coli lipids.

The molecular mechanism of cotranslational membrane protein recognition and targeting by SecA.

Cotranslational protein targeting is a conserved process for membrane protein biogenesis. In Escherichia coli, the essential ATPase SecA was found to cotranslationally target a subset of nascent membrane proteins to the SecYEG translocase at the plasma membrane. The molecular mechanism of this pathway remains unclear. Here we use biochemical and cryoelectron microscopy analyses to show that the amino-terminal amphipathic helix of SecA and the ribosomal protein uL23 form a composite binding site for the transmembrane domain (TMD) on the nascent protein. This binding mode further enables recognition of charged residues flanking the nascent TMD and thus explains the specificity of SecA recognition. Finally, we show that membrane-embedded SecYEG promotes handover of the translating ribosome from SecA to the translocase via a concerted mechanism. Our work provides a molecular description of the SecA-mediated cotranslational targeting pathway and demonstrates an unprecedented role of the ribosome in shielding nascent TMDs.

Two distinct anionic phospholipid-dependent events involved in SecA-mediated protein translocation.

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.

Molecular Mimicry of SecA and Signal Recognition Particle Binding to the Bacterial Ribosome.

Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome.IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time.