The long alpha-helix of SecA is important for the ATPase coupling of translocation

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.     

Global co-ordination of protein translocation by the SecA IRA1 switch

SecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain. We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1. IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e. DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover. IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation.     

Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.     

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.     

Identification of the preprotein binding domain of SecA

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.     

Two independent mechanisms down-regulate the intrinsic SecA ATPase activity

SecA initiates protein translocation by interacting with ATP, preprotein, and the SecYEG membrane components. Under such conditions, it undergoes a conformational change characterized as membrane insertion, which is then followed by hydrolysis of ATP, enabling the release of the preprotein and deinsertion of SecA itself for the next cycle of reactions. Without ongoing translocation, the ATPase activity of SecA is kept very low. Previously, it was shown that the C-terminal 34-kDa domain of SecA interacts with the N-terminal 68-kDa ATPase domain to down-regulate the ATPase. Here, we show, using a deregulated SecA mutant, that the intrinsic ATPase activity is subject to dual inhibitory mechanisms. Thus, the proposed second ATP-binding domain down-regulates the ATPase activity executed by the primary ATPase domain. This regulation, within the N-terminal ATPase domain, operates independently of the C-terminal domain-mediated regulation. The absence of both the mechanisms resulted in a 50-fold elevation of translocation-uncoupled ATP hydrolysis.     

Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain

SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219-244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.     

Identification of two interaction sites in SecY that are important for the functional interaction with SecA

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel.     

Monitoring the Activity of Single Translocons

Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc-reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Förster resonance energy transfer, we show for the first time that isolated nanodisc-reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co-reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc-reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Förster resonance energy transfer within the nanodisc-isolated monomeric SecYEG demonstrated a nucleotide-dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc-reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single-molecule analysis of dynamic aspects of protein translocation.     

Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA

The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.     

Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

A large conformational change couples the ATP binding site of SecA to the SecY protein channel

In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.     

Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function

SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs. We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controlling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its alpha-helical content. The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2-NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.     

Binding,activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase

The bacterial preprotein translocase is comprised of a membrane-embedded oligomeric SecYEG structure and a cytosolic dimeric SecA ATPase. The associations within SecYEG oligomers and SecA dimers, as well as between these two domains are dynamic and reversible. Here, it is shown that a covalently linked SecYEG dimer forms a functional translocase and a high affinity binding site for monomeric and dimeric SecA in solution. The interaction between these two domains stimulates the SecA ATPase, and nucleotides modulate the affinity and ratio of SecA monomers and dimers bound to the linked SecYEG complex. During the translocation reaction, the SecA monomer remains in stable association with a SecYEG protomer and the translocating preprotein. The nucleotides and translocation-dependent changes of SecA-SecYEG associations and the SecA dimeric state may reflect important facets of the preprotein translocation reaction.     

The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity.

SecYEG forms the protein-conducting channel of the Escherichia coli translocase. It binds the peripheral ATPase SecA that drives the preprotein translocation reaction. PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence. The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes. SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4. This activity correlates with an elevated SecA-translocation ATPase and SecA binding affinity. SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity. These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity.     

SecA dimer cross-linked at its subunit interface is functional for protein translocation

SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.     

Conformational state of the SecYEG-bound SecA probed by single tryptophan fluorescence spectroscopy

The SecYEG complex is a membrane-embedded channel that permits the passage of precursor proteins (preproteins) across the inner membrane of Escherichia coli. SecA is a molecular motor that associates with the SecYEG pore and drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the conformational state of soluble and SecYEG-bound SecA using single tryptophan mutants of SecA. The fluorescence spectral properties of the single tryptophans of SecA and their accessibility to the quencher acrylamide demonstrate that SecA undergoes a conformational change that results in a more compact structure upon binding of ATP and binding to the SecYEG pore. In addition, SecYEG-bound SecA undergoes ATP-dependent conformational changes that are not observed for soluble SecA. These data support a model in which binding to the SecYEG channel has a major impact on the SecA conformation.     

Topology of the SecA ATPase Bound to Large Unilamellar Vesicles

The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.     

The oligomeric state and arrangement of the active bacterial translocon

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.