Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA

In Escherichia coli , precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo , are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.     

Structural determinants of SecB recognition by SecA in bacterial protein translocation

SecB is a bacterial chaperone involved in directing pre-protein to the translocation pathway by its specific interaction with the peripheral membrane ATPase SecA. The SecB-binding site on SecA is located at its C terminus and consists of a stretch of highly conserved residues. The crystal structure of SecB in complex with the C-terminal 27 amino acids of SecA from Haemophilus influenzae shows that the SecA peptide is structured as a CCCH zinc-binding motif. One SecB tetramer is bound by two SecA peptides, and the interface involves primarily salt bridges and hydrogen bonding interactions. The structure explains the importance of the zinc-binding motif and conserved residues at the C terminus of SecA in its high-affinity binding with SecB. It also suggests a model of SecB-SecA interaction and its implication for the mechanism of pre-protein transfer in bacterial protein translocation.     

Identification of a Sequence Motif That Confers SecB Dependence on a SecB-Independent Secretory Protein In Vivo

SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.     

Orientation of SecA and SecB in complex, derived from disulfide cross-linking

SecA is the ATPase that acts as the motor for protein export in the general secretory, or Sec, system of Escherichia coli. The tetrameric cytoplasmic chaperone SecB binds to precursors of exported proteins before they can become stably folded and delivers them to SecA. During this delivery step, SecB binds to SecA. The complex between SecA and SecB that is maximally active in translocation contains two protomers of SecA bound to a tetramer of SecB. The aminoacyl residues on each protein that are involved in binding the other have previously been identified by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy; however, that study provided no information concerning the relative orientation of the proteins within the complex. Here we used our extensive collection of single-cysteine variants of the two proteins and subjected pairwise combinations of SecA and SecB to brief oxidation to identify residues in close proximity. These data were used to generate a model for the orientation of the two proteins within the complex.     

The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.

The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.     

Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR

Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translocase. Upon encounter, the signal peptide forms an alpha-helix that inserts into a flexible and elongated groove in SecA. The mode of binding is bimodal, with both hydrophobic and electrostatic interactions mediating recognition. The same groove is used by SecA to recognize a diverse set of signal sequences. Impairment of the signal-peptide binding to SecA results in significant translocation defects. The C-terminal tail of SecA occludes the groove and inhibits signal-peptide binding, but autoinhibition is relieved by the SecB chaperone. Finally, it is shown that SecA interconverts between two conformations in solution, suggesting a simple mechanism for polypeptide translocation.     

A thermodynamic coupling mechanism for the disaggregation of a model peptide substrate by chaperone secB

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated state(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precursor protein translocation machinery in Escherichia coli. We have investigated the interaction of the B-chain of insulin with chaperone SecB by light scattering, pyrene excimer fluorescence, and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B-chain of insulin. We show that SecB is capable of dissociating soluble B-chain aggregates as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B-chain aggregate by SecB has been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B-chains, rather it binds the small population of free B-chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate toward the individual B-chains. Thus SecB can rescue aggregated, partially folded/misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B-chain to chaperone SecB. The data suggests that the B-chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate.     

A mutation of Escherichia coli SecA protein that partially compensates for the absence of SecB.

The Escherichia coli SecB protein is a cytosolic chaperone protein that is required for rapid export of a subset of exported proteins. To aid in elucidation of the activities of SecB that contribute to rapid export kinetics, mutations that partially suppressed the export defect caused by the absence of SecB were selected. One of these mutations improves protein export in the absence of SecB and is the result of a duplication of SecA coding sequences, leading to the synthesis of a large, in-frame fusion protein. Unexpectedly, this mutation conferred a second phenotype. The secA mutation exacerbated the defective protein export caused by point mutations in the signal sequence of pre-maltose-binding protein. One explanation for these results is that the mutant SecA protein has sustained a duplication of its binding site(s) for exported protein precursors so that the mutant SecA is altered in its interaction with precursor molecules.     

SecB, a molecular chaperone with two faces

SecB is a molecular chaperone unique to the phylum Proteobacteria, which includes the majority of known Gram-negative bacteria of medical, industrial and agricultural significance. SecB is involved in the translocation of secretory proteins across the cytoplasmic membrane. The crystal structure of the Haemophilus influenzae SecB provides new insights into how SecB simultaneously recognizes its two ligands: unfolded preproteins and SecA, the ATPase subunit of the translocase. SecB uses its entire molecular surface for these two functions, but for preprotein release and its own membrane release, SecB relies on the catalytic activity of SecA. This defines SecB as a translocation-specific molecular chaperone.     

The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.     

Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.     

Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB.

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.     

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.     

The structural view of bacterial translocation-specific chaperone SecB: implications for function

SecB is a molecular chaperone that functions in bacterial post-translational protein translocation pathway. It maintains newly synthesized precursor polypeptide chains in a translocation-competent state and guides them to the translocon via its high-affinity binding to the ligand as well as to the membrane-embedded ATPase SecA. Recent advances in elucidating the structures of SecB have enabled the examination of protein function in the structural context. Structures of SecB from both Haemophilus influenzae and Escherichia coli support the early two-subsite polypeptide-binding model. In addition, the detailed molecular interaction between SecB and SecA was revealed by a structure of SecB in complex with the C-terminal zinc-containing domain of SecA. These observations explain the dual role of SecB plays in the translocation pathway, as a molecular chaperone and a specific targeting factor. A model of SecB-SecA complex suggests that the binding of SecA to SecB changes the conformation of the polypeptide binding sites in the chaperone, enabling transfer of precursor polypeptides from SecB to SecA. Recent studies also show the presence of a second zinc-independent SecB binding site in SecA and the new interaction might contribute to the function of SecB.     

Defining the role of the Escherichia coli chaperone SecB using comparative proteomics

To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by "classical" pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase.     

Zinc stabilizes the SecB binding site of SecA

The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a metal ion. Treatment of SecA with a zinc chelator resulted in a loss of the stimulatory effect of SecB on the SecA translocation ATPase activity, while the activity could be restored by the addition of ZnCl2. Interaction of SecB with the SecB binding domain of SecA is disrupted by chelators of divalent cations, and could be restored by the addition of Cu2+ or Zn2+. Atomic absorption and electrospray mass spectrometry revealed the presence of one zinc atom per monomeric carboxyl terminus of SecA. It is concluded that the SecB binding domain of SecA is stabilized by a zinc ion that promotes the functional binding of SecB to SecA.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.     

Kinetics and energetics of the translocation of maltose binding protein folding mutants

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.     

Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone

The evolutionarily conserved DnaJ proteins are essential components of Hsp70 chaperone systems. The DnaJ homologue of Escherichia coli associates with chaperone substrates and mediates their ATP hydrolysis-dependent locking into the binding cavity of its Hsp70 partner, DnaK. To determine the substrate specificity of DnaJ proteins, we screened 1633 peptides derived from 14 protein sequences for binding to E.coli DnaJ. The binding motif of DnaJ consists of a hydrophobic core of approximately eight residues enriched for aromatic and large aliphatic hydrophobic residues and arginine. The hydrophobicity of this motif explains why DnaJ itself can prevent protein aggregation. Although this motif shows differences from DnaK's binding motif, DnaJ and DnaK share the majority of binding peptides. In contrast to DnaK, DnaJ binds peptides consisting of L- and D-amino acids, and therefore is not restricted by backbone contacts. These features allow DnaJ to scan hydrophobic protein surfaces and initiate the functional cycle of the DnaK system by associating with hydrophobic exposed patches and subsequent targeting of DnaK to these or to hydrophobic patches in spatial neighbourhood.