Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
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ABSTRACT:
SUBMITTER: Robson A
PROVIDER: S-EPMC2653558 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Vibrios are a unique group of bacteria inhabiting a vast array of aquatic environments. Many Vibrio species are capable of infecting a wide assortment of hosts. Some of these species include V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. anguillarum, and V. cholerae. The ability of these organisms to utilize iron is essential in establishing both an infection in their hosts as well as surviving in the environment. Bacteria are able to sequester iron through the secretion of low molecular weight iron chelators termed siderophores. The iron-siderophore complexes are bound by specific outer membrane receptors and are brought through both the outer and inner membranes of the cell. The energy needed to drive this active transport is achieved through the TonB energy transduction system. When first elucidated in E. coli, the TonB system was shown to be a three protein complex consisting of TonB, ExbB and ExbD. Most Vibrio species carry two TonB systems. The second TonB system includes a fourth protein; TtpC, which is essential for TonB2 mediated iron transport. Some Vibrio species have been shown to carry a third TonB system that also includes a TtpC protein.
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Project description:Transduction of adenosine triphosphate (ATP) chemical-bond energy into work to drive large-scale conformational changes is common in proteins. Two specific examples of ATP-utilizing proteins are the nitrogenase iron protein and the ATP binding-cassette transporter protein, BtuCD. Nitrogenase catalyzes biological nitrogen fixation whereas BtuCD transports vitamin B(12) across membranes. Both proteins drive their reactions with ATP. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated in these proteins, a coarse-grained elastic network model is employed. The analysis shows that subunits of the proteins move against each other in a concerted manner. The lowest-frequency modes of the nitrogenase iron protein and of the ATP binding-cassette transporter BtuCD protein are found to link the functionally critical domains, and these modes are suggested to be responsible for (at least the initial stages) large-scale ATP-coupled conformational changes.
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Project description:UnlabelledThe TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them.ImportanceCanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.
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Project description:In bacteria, the membrane protein complex SecY/E/G and SecA ATPase are essential for protein translocation. About 30% of newly synthesized proteins in the cytosol are targeted to and translocated across the cytoplasmic membrane by the Sec factors. Although a number of single-molecule analyses and structural studies, including the crystal structure of SecYEG complexed with SecA, have been published, the underlying molecular mechanisms and the functional oligomer states remain elusive. In this study, we constructed a fusion protein SecY-SecA, which induces the formation of the SecY-A/SecE/SecG complex (SecYAEG), to enable investigation of the molecular mechanisms by advanced single-molecule analyses. SecYAEG-reconstituted liposomes were found to possess protein translocation activity in vitro and form stable intermediates capable of the translocation using a mutant substrate protein. We additionally found that one unit of SecYAEG complex embedded into a nanodisc, using membrane scaffold proteins, interacts strongly with the substrate. The isolated SecYAEG-reconstituted nanodisc is a promising tool for investigation of the molecular mechanisms by which a single unit of Sec machinery mediates protein translocation.
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Project description:SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ-benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X-ray structures and assessing their in vivo photo-crosslinking pattern. A discrete anti-parallel 1M6N-like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG-bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer-driven translocation mechanism.
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Project description:The mitochondrial ADP/ATP carrier, also called adenine nucleotide translocase, accomplishes one of the most important transport activities in eukaryotic cells, importing ADP into the mitochondrial matrix for ATP synthesis, and exporting ATP to fuel cellular activities. In the transport cycle, the carrier changes between a cytoplasmic and matrix state, in which the central substrate binding site is alternately accessible to these compartments. A structure of a cytoplasmic state was known, but recently, a structure of a matrix-state in complex with bongkrekic acid was solved. Comparison of the two states explains the function of highly conserved sequence features and reveals that the transport mechanism is unique, involving the coordinated movement of six dynamic elements around a central translocation pathway.
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Project description: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.
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Project description:Although the binding change mechanism of rotary catalysis by which F1-ATPase hydrolyzes ATP has been supported by equilibrium, kinetic, and structural observations, many questions concerning the function remain unanswered. Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology. Making use of the results of free energy simulations and experimental binding constant measurements, a model is developed for the free energy change during the hydrolysis cycle. This model makes possible the development of a kinetic scheme for ATP hydrolysis by F1-ATPase, in which the rate constants are associated with specific configurations of the beta subunits. An essential new element is that the strong binding site for ADP,Pi is shown to be the betaDP site, in contrast to the strong binding site for ATP, which is betaTP. This result provides a rationale for the rotation of the gamma subunit, which induces the cooperativity required for a tri-site binding change mechanism. The model explains a series of experimental data, including the ATP concentration dependence of the rate of hydrolysis and catalytic site occupation for both the Escherichia coli F1-ATPase (EcF1) and Thermophilic Bacillus PS3 F1-ATPase (TF1), which have different behavior.
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Project description:Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.
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Project description:Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study, we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger subdomain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger subdomain of SecA, and they are also consistent with the proposed role of this subdomain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of SecYEG protein. In addition, our work highlights the utility of this system to conveniently engineer and study the interaction of SecA with any signal peptide of interest as well as its potential use for X-ray crystallographic studies given issues with exogenous signal peptide solubility.
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