The SecB chaperone is involved in the secretion of the Serratia marcescens HasA protein through an ABC transporter.

The secretion pathways of the heme-binding protein HasA from Serratia marcescens and of the metalloproteases A, B, C and G from Erwinia chrysanthemi have been reconstituted in Escherichia coli. They are secreted in a single step from the cytoplasm across both membranes of the Gram-negative envelope, after recognition of their specific C-terminal secretion signal by their cognate ABC transporter. We report strong evidence that both HasA and the metalloproteases bind the SecB chaperone involved in the export of several envelope proteins via the Sec pathway. We also show that the secretion of the HasA protein is strongly dependent upon SecB in the reconstituted system, whereas that of the proteases is not. HasA secretion in the original host is strongly inhibited by a protein known to interfere with E.coli SecB function. We propose that the proteins secreted by the ABC pathway may have to be unfolded for efficient secretion.     

The SecB chaperone is bifunctional in Serratia marcescens: SecB is involved in the Sec pathway and required for HasA secretion by the ABC transporter

HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in Escherichia coli, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the secB gene from S. marcescens. We show that S. marcescens SecB is 93% identical to E. coli SecB and complements the secretion defects of a secB mutant of E. coli for both the Sec and ABC pathways of HasA secretion. In S. marcescens, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that S. marcescens SecB is the genuine chaperone for HasA secretion in S. marcescens. These results also demonstrate that S. marcescens SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of secB point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.     

The N terminus of the HasA protein and the SecB chaperone cooperate in the efficient targeting and secretion of HasA via the ATP-binding cassette transporter.

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.     

Antifolding activity of the SecB chaperone is essential for secretion of HasA,a quickly folding ABC pathway substrate

We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s(-1)) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s(-1)) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.     

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.     

NMR studies of the C-terminal secretion signal of the haem-binding protein, HasA.

HasA is a haem-binding protein which is secreted under iron-deficiency conditions by the gram-negative bacterium Serratia marcescens. It is a monomer of 19 kDa (187 residues) able to bind free haem as well as to capture it from haemoglobin. HasA delivers haem to a specific outer-membrane receptor HasR and allows the bacteria to grow in the absence of any other source of iron. It is secreted by a signal peptide-independent pathway which involves a C-terminal secretion signal and an ABC (ATP-binding cassette) transporter. The C-terminal region of the secretion signal containing the essential secretion motif is cleaved during or after the secretion process by proteases secreted by the bacteria. In this work, we study by 1H NMR the conformation of the C-terminal extremity of HasA in the whole protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environment. We identify a helical region followed by a random-coil C-terminus in the peptide-micelle complex and we show that in both the whole protein and the complex, the last 15 residues containing the motif essential for secretion are highly flexible and unstructured. This flexibility may be a prerequisite to the recognition of HasA by its ABC transporter. We determine the cleavage site of the C-terminal extremity of the protein and analyse the effect of the cleavage on the haem acquisition process.     

The crystal structure of the secreted dimeric form of the hemophore HasA reveals a domain swapping with an exchanged heme ligand

To satisfy their iron needs, several Gram-negative bacteria use a heme uptake system involving an extracellular heme-binding protein called hemophore. The function of the hemophore is to acquire free or hemoprotein-bound heme and to transfer it to HasR, its specific outer membrane receptor, by protein-protein interaction. The hemophore HasA secreted by Serratia marcescens, an opportunistic pathogen, was the first to be identified and is now very well characterized. HasA is a monomer that binds one b heme with strong affinity. The heme in HasA is highly exposed to solvent and coordinated by an unusual pair of ligands, a histidine and a tyrosine. Here, we report the identification, the characterization and the X-ray structure of a dimeric form of HasA from S. marcescens: DHasA. We show that both monomeric and dimeric forms are secreted in iron deficient conditions by S. marcescens. The crystal structure of DHasA reveals that it is a domain swapped dimer. The overall structure of each monomeric subunit of DHasA is very similar to that of HasA but formed by parts coming from the two different polypeptide chains, involving one of the heme ligands. Consequently DHasA binds two heme molecules by residues coming from both polypeptide chains. We show here that, while DHasA can bind two heme molecules, it is not able to deliver them to the receptor HasR. However, DHasA can efficiently transfer its heme to the monomeric form that, in turn, delivers it to HasR. We assume that DHasA can function as a heme reservoir in the hemophore system.     

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.     

Serratia ATP-binding cassette protein exporter, Lip, recognizes a protein region upstream of the C terminus for specific secretion

Serratia marcescens ATP-binding cassette (ABC) exporter, the Lip system, secretes lipase (LipA(SM)), metalloproteases, and a cell surface layer protein homologue but not a heme acquisition protein, HasA (HasA(SM)). Secretion of HasA(SM) is limited to the Has(SM) system. However, HasA proteins from Pseudomonas fluorescens (HasA(PF)) and Pseudomonas aeruginosa were exported through the Lip and Has(SM) systems. To investigate the specificity in Lip exporter-mediated secretion, secretion analysis was performed using chimeras containing the HasA(PF) and HasA(SM) sequences. The segment Val-Ala-Leu (designated R1 to R3 sites), which is present close to the C terminus of HasA(PF) but not HasA(SM), was revealed to be involved in the substrate specificity of the Lip exporter. Introduction of amino acid substitutions into the R1-R5 region demonstrated that R1, R3, R4, and R5 sites require some specific amino acid residues for Lip-mediated secretion. The amino acid sequence of the region was conserved considerably among the proteins secreted by the Lip exporter. On the contrary, the region was not related to HasA secretion through the Has(SM) system. Interestingly, a typical C-terminal motif, so far regarded as a secretion signal, was not necessary for secretion through either the Lip or the Has(SM) exporter. In LipA(SM) secretion via the Lip system, the typical C-terminal motif was not essential either, but the presence of a sequence similar to Val-Ala-Leu and its location from the C terminus greatly affect the secretion level. Secretion analyses using hybrid exporters and competitors exhibited that the R1-R5 region was recognized by an ABC protein of the Lip exporter, LipB, and that the mutations aborting Lip-mediated secretion in the region resulted in a loss of the affinity to LipB. Thus, a determinant within the secretory protein for Lip-mediated secretion was fully defined.     

Expression of Escherichia coli SecB in Bacillus subtilis facilitates secretion of the SecB-dependent maltose-binding protein of E. coli.

Less than 20% of the Escherichia coli maltose-binding protein (MBP) synthesized in Bacillus subtilis is exported. However, a portion of the secreted MBP was processed cotranslationally. Coexpression of SecB, a secretion-related chaperone of E. coli, stimulated posttranslational export of MBP in B. subtilis but inhibited its cotranslational processing. Export of a SecB-independent MBP-ribose-binding protein hybrid precursor was not enhanced by SecB. A slowly folding MBP derivative (MBP-Y283D) was more efficiently secreted than wild-type MBP, suggesting that the antifolding activity of SecB promotes posttranslational secretion of MBP in B. subtilis.     

Functional characterization of the HasA(PF) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor

Hemophores are secreted by several gram-negative bacteria (Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Yersinia pestis) and form a family of homologous proteins. Unlike the S. marcescens hemophore (HasA(SM)), the P. fluorescens hemophore HasA(PF) has an additional region of 12 residues located immediately upstream from the C-terminal secretion signal. We show that HasA(PF) undergoes a C-terminal cleavage which removes the last 21 residues when secreted from P. fluorescens and that only the processed form is able to deliver heme to the S. marcescens outer membrane hemophore-specific receptor, HasR(SM). Functional analysis of variants including those with an internal deletion of the extra C-terminal domain show that the secretion signal does not inhibit the biological activity, whereas the 12-amino-acid region located upstream does. This extra domain may inhibit the interaction of the hemophore with HasR(SM). To localize the hemophore regions involved in binding to HasR, chimeric HasA(PF)-HasA(SM) proteins were tested for biological activity. We show that residues 153 to 180 of HasA(PF) are necessary for its interaction with the receptor.     

革兰氏阴性菌血红素载体蛋白Hemophore的结构及作用机制

血红素作为宿主体内最丰富的铁离子来源,是致病菌营养竞争的主要目标,尤其对于血红素自身合成途径部分丧失的细菌。革兰氏阴性菌血红素转运系统由血红素载体蛋白(Hemophore)、外膜血红素受体、TonB-ExbB-ExbD复合物、ABC转运体等组成。Hemophore是存在于细菌细胞膜上或分泌到胞外环境中的一种蛋白。它能从宿主血红素结合蛋白中捕获血红素并将其传递给外膜受体。目前,在不同革兰氏阴性菌中已发现3种类型的Hemophore,分别是HasA、HxuA和HmuY型。本文将详细描述这3种Hemophore捕获血红素及与外膜受体相互作用的机制,以期为进一步研究其他细菌血红素载体蛋白的功能及作用机制奠定基础。     

Protein secretion by gram-negative bacterial ABC exporters

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, issec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins: an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide.Erwinia chrysanthemi metalloproteinases B and C, andSerratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on proteinase and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter/secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three component association was ordered and substrate binding was required for the formation of this multiprotein complex. Presented at the SymposiumRegulatory Aspects of Bacterial Cell Biology, Prague, October 16–17, 1996.     

Protein secretion by hybrid bacterial ABC-transporters: specific functions of the membrane ATPase and the membrane fusion protein.

The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous although the secreted polypeptides share no sequence homology. Whereas protease C can use both translocators, HasA is secreted only by its specific transporter. Functional analysis of protease C and HasA secretion through hybrid transporters obtained by combining components from each system demonstrates that the ABC-protein is responsible for the substrate specificity and that inhibition of protease C secretion in the presence of HasA results from a defective interaction between HasA and the ABC-protein. We also show that the outer membrane protein, TolC, can combine with the membrane fusion protein HasE in the presence of either ABC-protein to form a functional transporter but not with the membrane fusion protein, PrtE. This indicates a specific interaction between the outer membrane component and the membrane fusion protein.     

The heme transfer from the soluble HasA hemophore to its membrane-bound receptor HasR is driven by protein-protein interaction from a high to a lower affinity binding site

HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 x 10(10) m(-1)). Heme is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 x 10(6) m(-1) and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA.HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.     

The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro.

It has been proposed that the cytoplasmic SecB protein functions as a component of the Escherichia coli protein export machinery by serving as an antifolding factor that retards folding of the precursor maltose-binding protein (preMBP) into a translocation-incompetent form. In this study, it was found that SecB directly interacts with wild-type preMBP and various mutationally altered MBP species synthesized in vitro to form a SecB-MBP complex that can be precipitated with anti-SecB serum. The association of SecB with wild-type preMBP was relatively unstable; such a complex was formed only when SecB was present cotranslationally or after denaturation of previously synthesized preMBP and was detected with only low efficiency. In marked contrast, MBP species that were defective in the ability to assume the stable conformation of wild-type preMBP or that exhibited significantly slower folding kinetics formed much more stable complexes with SecB. In one case, we demonstrated that SecB did not need to be present cotranslationally for complex formation to occur. Formation of a complex between SecB and MBP was clearly not dependent on the MBP signal peptide. However, we were unable to detect complex formation between SecB and MBP lacking virtually the entire signal peptide but having a completely intact mature moiety. This MBP species folded at a rate considerably faster than that of wild-type preMBP. The propensity of this mutant protein to assume the native conformation of mature MBP apparently precludes a stable association with SecB, whereas an MBP species lacking a signal peptide but exhibiting altered folding properties did form a complex with SecB that could be precipitated with anti-SecB serum.     

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.     

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.     

A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli.

The utilization by Serratia marcescens of heme bound to hemoglobin requires HasA, an extracellular heme-binding protein. This unique heme acquisition system was studied in an Escherichia coli hemA mutant that was a heme auxotroph. We identified a 92-kDa iron-regulated S. marcescens outer membrane protein, HasR, which alone enabled the E. coli hemA mutant to grow on heme or hemoglobin as a porphyrin source. The concomitant secretion of HasA by the HasR-producing hemA mutant greatly facilitates the acquisition of heme from hemoglobin. This is the first report of a synergy between an outer membrane protein and an extracellular heme-binding protein, HasA, acting as a heme carrier, which we termed a hemophore.     

Histidine pK(a) shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasA(SM)

The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo- and holo-forms of HasA(SM) in solution. Holo-HasA(SM) was formed with gallium-protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side-chain (15)N and (1)H resonances of the six HasA(SM) histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.