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.     

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.     

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.     

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.     

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.     

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.     

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.     

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.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

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.     

Interdependent Effects of the Charge of the N-Terminal Region of the Signal Peptide,SecA, and SecB on Secretion of Alkaline Phosphatase in Escherichia coli

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Replacement of positively charged Lys(–20) with uncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.     

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.     

Crystal structure of the bacterial protein export chaperone secB

SecB is a bacterial molecular chaperone involved in mediating translocation of newly synthesized polypeptides across the cytoplasmic membrane of bacteria. The crystal structure of SecB from Haemophilus influenzae shows that the molecule is a tetramer organized as a dimer of dimers. Two long channels run along the side of the molecule. These are bounded by flexible loops and lined with conserved hydrophobic amino acids, which define a suitable environment for binding non-native polypeptides. The structure also reveals an acidic region on the top surface of the molecule, several residues of which have been implicated in binding to SecA, its downstream target.     

Interaction of effect on secretion of alkaline phosphatase from E. coli by charge of the N-terminal part of the signal peptide and proteins SecB and SecA

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Substitution of positively charged Lys(-20) with noncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase (PhoA) precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.     

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.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Complexes between protein export chaperone SecB and SecA. Evidence for separate sites on SecA providing binding energy and regulatory interactions

During localization to the periplasmic space or to the outer membrane of Escherichia coli some proteins are dependent on binding to the cytosolic chaperone SecB, which in turn is targeted to the membrane by specific interaction with SecA, a peripheral component of the translocase. Five variant forms of SecB, previously demonstrated to be defective in mediating export in vivo (Gannon, P. M., and Kumamoto, C. A. (1993) J. Biol. Chem. 268, 1590-1595; Kimsey, H. K., Dagarag, M. D., and Kumamoto, C. A. (1995) J. Biol. Chem. 270, 22831-22835) were investigated with respect to their ability to bind SecA both in solution and at the membrane translocase. We present evidence that at least two regions of SecA are involved in the formation of active complexes with SecB. The variant forms of SecB were all capable of interacting with SecA in solution to form complexes with stability similar to that of complexes between SecA and wild-type SecB. However, the variant forms were defective in interaction with a separate region of SecA, which was shown to trigger a change that was correlated to activation of the complex. The region of SecA involved in activation of the complexes was defined as the extreme carboxyl-terminal 21 aminoacyl residues.     

Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein.

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.     

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.     

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.