ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila

Aeromonas hydrophila uses the type II secretion system (T2SS) to transport protein toxins across the outer membrane. The inner membrane complex ExeAB is required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan‐binding domain (Pfam number PF01471) conserved in many peptidoglycan‐related proteins is present in the periplasmic region of ExeA (P‐ExeA). In this study, co‐sedimentation analysis revealed that P‐ExeA was able to bind to highly pure peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in native PAGE, gel filtration and cross‐linking experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at 30°C and above. These results provide evidence that the putative peptidoglycan‐binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions facilitate the multimerization of ExeA, favouring a model in which the protein forms a multimeric structure on the peptidoglycan during the ExeAB‐dependent assembly of the secretin multimer in the outer membrane.     

Interactions between peptidoglycan and the ExeAB complex during assembly of the type II secretin of Aeromonas hydrophila

Aeromonas hydrophila transports extracellular protein toxins via the type II secretion system, an export mechanism comprised of numerous proteins that spans both the inner and outer membranes. Two components of this secretion system, ExeA and ExeB, form a complex in the inner membrane that functions to locate and/or assemble the ExeD secretin in the outer membrane. In the studies reported here, two-codon insertion mutagenesis of exeA revealed that an insertion at amino acid 495 in the C-terminal region of ExeA did not alter ExeAB complex formation yet completely abrogated its involvement in ExeD secretin assembly and thus rendered the bacteria secretion negative. In silico analysis of protein motifs with similar amino acid profiles revealed that this amino acid is located within a putative peptidoglycan (PG) binding motif in the periplasmic domain of ExeA. Substitution mutations of three highly conserved amino acids in the motif were constructed. In cells expressing each of these mutants, the ability to assemble the ExeD secretin or secrete aerolysin was lost, while ExeA retained the ability to form a complex with ExeB. In in vivo cross-linking experiments, wild-type ExeA could be cross-linked to PG, whereas the three substitution mutants of ExeA could not. These data indicate that PG binding and/or remodelling plays a role in the function of the ExeAB complex during assembly of the ExeD secretin.     

Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species

The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila, GspA(Ah), is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB(Ah)). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD(Ah)) secretin multimer in the outer membrane. In this study, the requirement for GspAB in the assembly of the T2SS secretin in Aeromonas and Vibrio species was investigated. We have demonstrated a requirement for GspAB in T2SS assembly in Aeromonas salmonicida, similar to that previously observed in A. hydrophila. In the Vibrionaceae species Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus, gspA mutations significantly decreased assembly of the secretin multimer but had minimal effects on the secretion of T2SS substrates. The lack of effect on secretion of the mutant of gspA of V. cholerae (gspA(Vc)) was explained by the finding that native secretin expression greatly exceeds the level needed for efficient secretion in V. cholerae. In cross-complementation experiments, secretin assembly and secretion in an A. hydrophila gspA mutant were partially restored by the expression of GspAB from V. cholerae in trans, further suggesting that GspAB(Vc) performs the same role in Vibrio species as GspAB(Ah) does in the aeromonads. These results indicate that the GspAB complex is functional in the assembly of the secretin in Vibrio species but that a redundancy of GspAB function may exist in this genus.     

Peptidoglycan recognition by Pal, an outer membrane lipoprotein

Peptidoglycan-associated lipoprotein (Pal) is a potential vaccine candidate from Haemophilus influenzae that is highly conserved in Gram-negative bacteria and anchored to the outer membrane through an N-terminal lipid attachment. Pal stabilizes the outer membrane by providing a noncovalent link to the peptidoglycan (PG) layer through a periplasmic domain. Using NMR spectroscopy, we determined the three-dimensional structure of a complex between the periplasmic domain of Pal and a biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-alpha-d-Glu-m-Dap-D-Ala-d-Ala (m-Dap is meso-diaminopimelate). Pal has a binding pocket lined with conserved surface residues that interacts exclusively with the peptide portion of the ligand. The m-Dap residue, which is mainly found in the cell walls of Gram-negative bacteria, is sequestered in this pocket and plays an important role by forming hydrogen bond and hydrophobic contacts to Pal. The structure provides insight into the mode of cell wall recognition for a broad class of Gram-negative membrane proteins, including OmpA and MotB, which have peptidoglycan-binding domains homologous to that of Pal.     

Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD

The type two secretion system is a large, trans-envelope apparatus that secretes toxins across the outer membrane of many Gram-negative bacteria. In Aeromonas hydrophila, ExeA interacts with peptidoglycan and forms a heteromultimeric complex with ExeB that is required for assembly of the ExeD secretin of the secretion system in the outer membrane. While the peptidoglycan-ExeAB (PG-AB) complex is required for ExeD assembly, the assembly mechanism remains unresolved. We analyzed protein-protein interactions to address the hypothesis that ExeD assembly in the outer membrane requires direct interaction with the PG-AB complex. Yeast and bacterial two hybrid analyses demonstrated an interaction between the periplasmic domains of ExeB and ExeD. Two-codon insertion mutagenesis of exeD disrupted lipase secretion, and immunoblotting of whole cells demonstrated significantly reduced secretin in mutant cells. Mapping of the two-codon insertions and deletion analysis showed that the ExeB-ExeD interaction involves the N0 and N1 subdomains of ExeD. Rotational anisotropy using the purified periplasmic domains of ExeB and ExeD determined that the apparent dissociation constant of the interaction is 1.19±0.16 µM. These results contribute important support for a putative mechanism by which the PG-AB complex facilitates assembly of ExeD through direct interaction between ExeB and ExeD. Furthermore, our results provide novel insight into the assembly function of ExeB that may contribute to elucidating the role of homologous proteins in secretion of toxins from other Gram negative pathogens.     

A TonB-like protein and a novel membrane protein containing an ATP-binding cassette function together in exotoxin secretion

Protein secretion by many Gram-negative bacteria occurs via the type II pathway involving translocation across the cytoplasmic and outer membranes in separate steps. The mechanism by which metabolic energy is supplied to the translocation across the outer membrane is unknown. Here we show that two Aeromonas hydrophila inner membrane proteins, ExeA and ExeB, are required for this process. ExeB bears sequence as well as topological similarity to TonB, a protein which opens gated ports for the inward translocation of ligands across the outer membrane. ExeA is a novel membrane protein which contains a consensus ATP-binding site. Mutations in this site dramatically decreased the rate of secretion of the toxin aerolysin from the cell. ExeB was stable when overproduced in the presence of ExeA, but was degraded when synthesized in its absence, indicating that the two proteins form a complex. These results suggest that ExeA and ExeB may act together to transduce metabolic energy to the opening of a secretion port in the outer membrane.     

Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram-negative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N-terminal domain and a protease-resistant C-terminal domain that remains multimeric after proteolysis. The stable C-terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C-terminal end to which the secretin-specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N-terminal domain folds back into the large cavity of the channel that is formed by the C-terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.     

The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain,a new peptidoglycan binding domain

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram‐negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N‐acetylmuramyl‐L‐alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a β‐sandwich of two symmetrical four‐stranded β‐sheets exposing highly conserved motifs on the two outer faces. We show that this N‐terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan‐binding domain. The C‐terminal catalytic domain shows an auto‐inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity.     

Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges

ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity. The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The 1.75 angstroms crystal structure of the targeting domain shows an all-beta fold similar to typical SH3s but with unique features. The structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.     

Purification and characterization of the N-terminal domain of ExeA: a novel ATPase involved in the type II secretion pathway of Aeromonas hydrophila

Aeromonas hydrophila secretes a number of degradative enzymes and toxins into the external milieu via the type II secretory pathway or secreton. ExeA is an essential component of this system and is necessary for the localization and/or multimerization of the secretin ExeD. ExeA contains two sequence motifs characteristic of the Walker superfamily of ATPases. Previous examination of substitution derivatives altered in these motifs suggested that ATP binding or hydrolysis is required for ExeAB complex formation and subsequent secretion function. To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity and anion-exchange chromatographic techniques. Purified preparations of cytExeA exhibited ATPase activity in the presence of several divalent cations, Mg2+ being the preferred cation, with an optimum reaction temperature of approximately 37 to 42 degrees C and an optimum pH of 7 to 8. cytExeA exhibited an apparent K(m) for Mg-ATP of 0.22 mM and a V(max) of 0.72 nmol min(-1) mg(-1) of protein. cytExeA displayed low specificity for nucleoside triphosphate substrates and was significantly inhibited by F-type ATPase inhibitors. Gel filtration analyses of cytExeA, ExeA, and ExeAB indicated that ExeA dimerizes and forms a very large complex with ExeB. These findings support a model whereby ExeAB utilizes energy derived from ATP hydrolysis to facilitate the correct localization and multimerization of the ExeD secretin.     

Species-specific functioning of the Pseudomonas XcpQ secretin: role for the C-terminal homology domain and lipopolysaccharide

Secretins are oligomeric proteins that mediate the export of macromolecules across the bacterial outer membrane. The members of the secretin superfamily possess a C-terminal homology domain that is important for oligomerization and channel formation, while their N-terminal halves are thought to be involved in system-specific interactions. The XcpQ secretin of Pseudomonas spp. is a component of the type II secretion pathway. XcpQ from Pseudomonas alcaligenes is not able to functionally replace the secretin of the closely related species Pseudomonas aeruginosa. By analysis of chimeric XcpQ proteins, a region important for species-specific functioning was mapped between amino acid residues 344 and 478 in the C-terminal homology domain. Two chromosomal suppressor mutations were obtained that resulted in the proper functioning in P. aeruginosa of P. alcaligenes XcpQ and inactive hybrids. These mutations caused a defect in the synthesis of the lipopolysaccharide (LPS) outer core region. Subsequent analysis of different LPS mutants showed that changes in the outer core and not the loss of O antigen caused the suppressor phenotype. High concentrations of divalent cations in the growth medium also allowed P. alcaligenes XcpQ and inactive hybrids to function properly in P. aeruginosa. Since divalent cations are known to affect the structure of LPS, this observation supports the hypothesis that LPS has a role in the functioning of secretins.     

Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations.

Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product. We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions. The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features. Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity. Mutations in the helical domain retain ATP binding activity. Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation. We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations.     

Outer membrane targeting of secretin PulD protein relies on disordered domain recognition by a dedicated chaperone

Interaction of bacterial outer membrane secretin PulD with its dedicated lipoprotein chaperone PulS relies on a disorder-to-order transition of the chaperone binding (S) domain near the PulD C terminus. PulS interacts with purified S domain to form a 1:1 complex. Circular dichroism, one-dimensional NMR, and hydrodynamic measurements indicate that the S domain is elongated and intrinsically disordered but gains secondary structure upon binding to PulS. Limited proteolysis and mass spectrometry identified the 28 C-terminal residues of the S domain as a minimal binding site with low nanomolar affinity for PulS in vitro that is sufficient for outer membrane targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization and does not interact with PulS, but it is required for secretin function in type II secretion. Although other secretin chaperones differ substantially from PulS in sequence and secondary structure, they have all adopted at least superficially similar mechanisms of interaction with their cognate secretins, suggesting that intrinsically disordered regions facilitate rapid interaction between secretins and their chaperones.     

Structure and biochemical analysis of a secretin pilot protein

The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 A the structure of MxiM28-142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525-570, hinders lipid binding to MxiM.     

Mutational Analysis of CvaA in the Highly Conserved Domain of the Membrane Fusion Protein Family

The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue–hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV. Received: 6 February 1999 / Accepted: 26 June 1999     

Structural and functional analysis of phosphothreonine-dependent FHA domain interactions

FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.     

Quinone binding and reduction by respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.     

Structural origins of aminoglycoside specificity for prokaryotic ribosomes

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.     

Crystal structure of outer membrane protein NMB0315 from Neisseria meningitidis

NMB0315 is an outer membrane protein of Neisseria meningitidis serogroup B (NMB) and a potential candidate for a broad-spectrum vaccine against meningococcal disease. The crystal structure of NMB0315 was solved by single-wavelength anomalous dispersion (SAD) at a resolution of 2.4 Å and revealed to be a lysostaphin-type peptidase of the M23 metallopeptidase family. The overall structure consists of three well-separated domains and has no similarity to any previously published structure. However, only the topology of the carboxyl-terminal domain is highly conserved among members of this family, and this domain is a zinc-dependent catalytic unit. The amino-terminal domain of the structure blocks the substrate binding pocket in the carboxyl-terminal domain, indicating that the wild-type full-length protein is in an inactive conformational state. Our studies improve the understanding of the catalytic mechanism of M23 metallopeptidases.