Characterization of three areas of interactions stabilizing complexes between SecA and SecB, two proteins involved in protein export

The general secretory, Sec, system translocates precursor polypeptides from the cytosol across the cytoplasmic membrane in Escherichia coli. SecB, a small cytosolic chaperone, captures the precursor polypeptides before they fold and delivers them to the membrane translocon through interactions with SecA. Both SecB and SecA display twofold symmetry and yet the complex between the two is stabilized by contacts that are distributed asymmetrically. Two distinct regions of interaction have been defined previously and here we identify a third. Calorimetric studies of complexes stabilized by different subsets of these interactions were carried out to determine the binding affinities and the thermodynamic parameters that underlie them. We show here that there is no change in affinity when either one of two contact areas out of the three is lacking. This fact and the asymmetry of the binding contacts may be important to the function of the complex in protein export.     

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.     

Asymmetric binding between SecA and SecB two symmetric proteins: implications for function in export

SecB, a small tetrameric chaperone in Escherichia coli, facilitates export of precursor polypeptides from the cytoplasm to the periplasmic space. During this process, SecB displays two modes of binding. As a chaperone, it binds promiscuously to precursors to maintain them in a non-native conformation. SecB also demonstrates specific recognition of, and binding to, SecA. SecB with the precursor tightly bound enters an export-active complex with SecA and must pass the ligand to SecA at the translocon in the membrane. Here we use variants of SecA and SecB to further probe these interactions. We show that, unexpectedly, the binding between the two symmetric molecules is asymmetric and that the C-terminal alpha-helices of SecB bind in the interfacial region of the SecA dimer. We suggest that disruption of this interface by SecB facilitates conformational changes of SecA that are crucial to the transfer of the precursor from SecB to SecA.     

SecA folding kinetics: a large dimeric protein rapidly forms multiple native states

SecA, a 202 kDa dimeric protein, is the ATPase for the Sec-dependent translocase of precursor proteins in vivo. SecA must undergo conformational changes, which may involve dissociation into a monomer, as it translocates the precursor protein across the inner membrane. To better understand the dynamics of SecA in vivo, protein folding studies to probe the native, intermediate, and unfolded species of SecA in vitro have been done. SecA folds through a stable dimeric intermediate and dimerizes in the dead-time of a manual-mixing kinetic experiment ( approximately 5-7 seconds). Here, stopped-flow fluorescence and CD, as well as ultra-rapid continuous flow fluorescence techniques, were used to further probe the rapid folding kinetics of SecA. In the absence of urea, rapid, near diffusion-limited ( approximately 10(9)M(-1)s(-1)) SecA dimerization occurs following a rate-limiting unimolecular rearrangement of a rapidly formed intermediate. Multiple kinetic folding and unfolding phases were observed and SecA was shown to have multiple native and unfolded states. Using sequential-mixing stopped-flow experiments, SecA was determined to fold via parallel channels with sequential intermediates. These results confirm that SecA is a highly dynamic protein, consistent with the rapid, major conformational changes it must undergo in vivo.     

The ATPase domain of SecA can form a tetramer in solution.

Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)=[monomer](4)/[tetramer]) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore.     

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane‐embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N‐terminal residues 2 through 11 of SecA and the C‐terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.     

Expression, purification, and characterization of Pseudomonas aeruginosa SecA

A secA gene from Pseudomonas aeruginosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13) under conditions where E. coli SecA was depleted. The binding of P. aeruginosa SecA (PaSecA) to the SP-Sepharose column was facilitated by ammonium sulfate fractionation but was not necessary for E. coli SecA (EcSecA) as the later bound more efficiently. PaSecA and EcSecA were purified by the single chromatographic step to greater than 98% purity and had a recovery of more than 20 and 40%, respectively, from the soluble fraction. This simple step purification obtained a higher homogeneity than previously reported. Cross-reactivity by immunoblotting showed that the purified PaSecA contained little EcSecA if any. The purified PaSecA is a dimer in solution, as judged by size exclusion chromatography, and is slightly larger than its counterpart EcSecA with an estimated molecular weight of 240 kDa. Further studies by the sedimentation velocity method indicate that PaSecA tends to remain as a monomer in solution. The purified PaSecA possessed ATPase activity; the intrinsic and liposome-stimulated ATPase specific activities of PaSecA were approximately 50% of EcSecA.     

Mapping of the docking of SecA onto the chaperone SecB by site-directed spin labeling: insight into the mechanism of ligand transfer during protein export

Export of protein into the periplasm of Escherichia coli via the general secretory system is achieved by action of a ternary complex comprising the polypeptide ligand, the chaperone SecB and SecA, a peripheral component of the membrane translocon, which is itself an ATPase. The unfolded ligand is captured initially by SecB and must be transferred to SecA and subsequently through the membrane translocon into the periplasm. We have taken the first steps in the elucidation of the mechanism of this dynamic transfer by determining the interface of interaction between SecB and SecA. Site-directed spin labeling and electron paramagnetic resonance spectroscopy were combined to identify which of the residues on SecB showed changes in spectral line shape upon addition of SecA. In all, 43% of the surface of SecB was covered by the 41 positions examined. A model of docking between SecB and SecA is proposed based on the pattern of amino acid residues on SecB shown to make contacts when in complex with SecA. This model in combination with previously published biochemical data provides insight into the transfer of the unfolded polypeptide from the chaperone SecB to SecA.     

Penetration into membrane of amino‐terminal region of SecA when associated with SecYEG in active complexes

The general secretory (Sec) system of Escherichia coli translocates both periplasmic and outer membrane proteins through the cytoplasmic membrane. The pathway through the membrane is provided by a highly conserved translocon, which in E. coli comprises two heterotrimeric integral membrane complexes, SecY, SecE, and SecG (SecYEG), and SecD, SecF, and YajC (SecDF/YajC). SecA is an associated ATPase that is essential to the function of the Sec system. SecA plays two roles, it targets precursors to the translocon with the help of SecB and it provides energy via hydrolysis of ATP. SecA exists both free in the cytoplasm and integrally membrane associated. Here we describe details of association of the amino‐terminal region of SecA with membrane. We use site‐directed spin labelling and electron paramagnetic resonance spectroscopy to show that when SecA is co‐assembled into lipids with SecYEG to yield highly active translocons, the N‐terminal region of SecA penetrates the membrane and lies at the interface between the polar and the hydrophobic regions, parallel to the plane of the membrane at a depth of approximately 5 Å. When SecA is bound to SecYEG, preassembled into proteoliposomes, or nonspecifically bound to lipids in the absence of SecYEG, the N‐terminal region penetrates more deeply (8 Å). Implications of partitioning of the SecA N‐terminal region into lipids on the complex between SecB carrying a precursor and SecA are discussed.     

Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.     

Sites of interaction between SecA and the chaperone SecB, two proteins involved in export

SecB, a small tetrameric cytosolic chaperone in Escherichia coli, facilitates the export of precursor poly-peptides by maintaining them in a nonnative conformation and passing them to SecA, which is a peripheral member of the membrane-bound translocation apparatus. It has been proposed by several laboratories that as SecA interacts with various components along the export pathway, it undergoes conformational changes that are crucial to its function. Here we report details of molecular interactions between SecA and SecB, which may serve as conformational switches. One site of interaction involves the final C-terminal 21 amino acids of SecA, which are positively charged and contain zinc. The C terminus of each subunit of the SecA dimer makes contact with the flat beta-sheet that is formed by each dimer of the SecB tetramer. Here we demonstrate that a second interaction exists between the extreme C-terminal alpha-helix of SecB and a site on SecA, as yet undefined but different from the C terminus of SecA. We investigated the energetics of the interactions by titration calorimetry and characterized the hydrodynamic properties of complexes stabilized by both interactions or each interaction singly using sedimentation velocity centrifugation.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

Structural determinants of protein translocation in bacteria: conformational flexibility of SecA IRA1 loop region

Bacteria employ the SecA motor protein to push unfolded proteins across the cytoplasmic membrane through the SecY protein‐conducting channel complex. The crystal structure of the SecA–SecY complex shows that the intramolecular regulator of ATPase1 (IRA1) SecA domain, made up of two helices and the loop between them, is partly inserted into the SecY conducting channel, with the loop between the helices as the main functional region. A computational analysis suggested that the entire IRA1 domain is structurally autonomous, and was the basis to synthesize peptide analogs of the SecA IRA1 loop region, to the aim of investigating its conformational preferences. Our study indicates that the loop region populates a predominantly flexible state, even in the presence of structuring agent. This provides indirect evidence that the SecA loop–SecY receptor docking involves loop‐mediated opening of the SecY channel. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery of the first SecA inhibitors using structure-based virtual screening

Bacterial protein secretion is a critical and complex process. The Sec machinery provides a major pathway for protein translocation across and integration into the cellular membrane in bacteria. Small molecule probes that perturb the functions of individual member proteins within the Sec machinery will be very important research tools as well as leads for future antimicrobial agent development. Herein we describe the discovery of inhibitors, through virtual screening, that specifically act on SecA ATPase, which is a critical member of the Sec system. These are the very first inhibitors reported for intrinsic SecA ATPase.     

Influence of different assignment conditions on the determination of symmetric homodimeric structures with ARIA

The ambiguous restraint for iterative assignment (ARIA) approach for NMR structure calculation is evaluated for symmetric homodimeric proteins by assessing the effect of several data analysis and assignment methods on the structure quality. In particular, we study the effects of network anchoring and spin-diffusion correction. The spin-diffusion correction improves the protein structure quality systematically, whereas network anchoring enhances the assignment efficiency by speeding up the convergence and coping with highly ambiguous data. For some homodimeric folds, network anchoring has been proved essential for unraveling both chain and proton assignment ambiguities.     

Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.     

Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon

Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.     

Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design

Candidatus Liberibacter asiaticus is the causal agent of Huanglongbing (HLB) disease of citrus. Current management practices have not been able to control HLB and stop the spread of HLB. The current study is focused on screening small molecule inhibitors against SecA protein of Ca. L. asiaticus. Homology modeling, structure based virtual screening and molecular docking methods have been used to find the novel inhibitory compounds against SecA activity at ATP binding region. At 20 μm 17 compounds showed >50% inhibition and four compounds had more than 65% inhibition. The most active compound has IC₅₀ value of 2.5 μM. The differences between the activities of the compounds are explained by their inter-molecular interactions at ATP binding site.     

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.     

以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

Sec途径(即分泌途径secretion pathway)是蛋白质转运的主要途径.其中,最为关键的组分之一是SecAATP酶,是蛋白质转运途径中的"动力泵",通过ATP的水解循环驱使蛋白质前体穿过细菌内膜,在细菌中是不可缺少的.我们推测抑制SecAATP酶活性的化合物.必然会在一定程度上抑制蛋白质的转运和分泌.通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用,利用本实验室构建的高效表达SecA蛋白的基因工程菌,建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型.利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系,验证了所建立的细胞水平筛选模型具有一定的特异性.研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性,值得进行深入的研究.