Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter

Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins.     

The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC

TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.     

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.     

Thermodynamic aspects of DsbD-mediated electron transport

DsbD from Escherichia coli transports electrons from cytoplasmic thioredoxin across the inner membrane to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of three domains: a periplasmic N-terminal domain, a central transmembrane domain (tmDsbD) and a periplasmic C-terminal domain. Each domain contains two essential cysteine residues that are required for electron transport. In contrast to the quinone reductase DsbB, HPLC analysis of the methanol/hexane extracts of purified DsbD revealed no presence of quinones, suggesting that the tmDsbD interacts with thioredoxin and the periplasmic C-terminal domain exclusively via disulfide exchange. We also demonstrate that a DsbD variant containing only the redox-active cysteine pair C163 and C285 in tmDsbD, reconstituted into liposomes, has a redox potential of − 0.246 V. The results show that all steps in the DsbD-mediated electron flow are thermodynamically favorable.     

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal ‘Peptidase_M75’ domain of 251 residues. The C-terminal domain contains a highly conserved ‘HXXE’ motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or ‘Psyr_3370’) encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M_W 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly α-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6 Å. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.     

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1–171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180–325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA^180–325). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA^180–325 with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA^1–325) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation.     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c₅₅₀ expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane β-barrel channel, an internal plug domain that occludes the channel, and globular N- and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an α-helix that caps the plug domain, or the N- and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and α-helix function to modulate the gating activity of the PapC twin pore.     

Cloning,sequence analysis and expression in E. coli of the DNA polymerase I gene from Chloroflexus aurantiacus,a green: nonsulfur eubacterium

We have cloned and sequenced the polA gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium, and expressed the recombinant protein in Escherichia coli. One open reading frame encodes a protein with 942 amino acids showing 38% identity with DNA polymerase I from E. coli. Sequence alignments with other members of DNA polymerase family A and analysis of the separate domains show that the central 3′-5′ exonuclease domain is 30% identical to the corresponding E. coli domain and that three sequence motifs associated with 3′-5′ exonuclease activity are conserved. Also, a protein fraction from E. coli expressing the Chloroflexus polymerase contains a thermostable 3′-5′ exonucleolytic activity, indicating that this activity is present in the enzyme, in agreement with the sequence analysis. The N-terminal 5′-3′ exonuclease domain and the C-terminal polymerase domain show 31 and 46% identity, respectively, with the corresponding E. coli domains and all sequence motifs associated with these two enzymatic activities also are conserved. Since several DNA polymerase I enzymes lack the proofreading activity associated with the central domain it has been suggested that the ancestral polA gene contained only the two more conserved N- and C-terminal domains and that the proofreading 3′-5′ exonuclease domain was introduced later in those eubacterial branches that have this activity. Our data indicate a different scenario where the ancestral polA gene contained both the exonucleolytic activities in addition to the polymerase activity and where several eubacterial branches lost the polymerase-associated proofreading activity during evolution.     

Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants

Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.     

Septal Localization of FtsQ, an Essential Cell Division Protein in Escherichia coli

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.     

Crystal structure and biochemical features of EfeB/YcdB from Escherichia coli O157: ASP235 plays divergent roles in different enzyme-catalyzed processes

EfeB/YcdB is a member of the dye-decolorizing peroxidase (DyP) protein family. A recent study has shown that this protein can extract iron from heme without breaking the tetrapyrrole ring. We report the crystal structure of EfeB from Escherichia coli O157 bound to heme at 1.95 Å resolution. The EfeB monomer contains two domains. The heme molecule is located in a large hydrophobic pocket in the C-terminal domain. A long loop connecting the two domains extensively interacts with the heme, which is a distinctive structural feature of EfeB homologues. A large tunnel formed by this loop and the β-sheet of C-terminal domain provides a potential cofactor/substrate binding site. Biochemical data show that the production of protoporphyrin IX (PPIX) is closely related to the peroxidation activity. The mutant D235N keeps nearly the same activity of guaiacol peroxidase as the wild-type protein, whereas the corresponding mutation in the classic DyP protein family completely abolished the peroxidation activity. These results suggest that EfeB is a unique member of the DyP protein family. In addition, dramatically enhanced fluorescence excitation and emission of EfeB-PPIX was observed, implying this protein may be used as a red color fluorescence marker.     

Molecular Dissection of Phage Endolysin: AN INTERDOMAIN INTERACTION CONFERS HOST SPECIFICITY IN LYSIN A OF MYCOBACTERIUM PHAGE D29*

Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages.     

Structure and Function of the Escherichia coli Tol-Pal Stator Protein TolR

TolR is a 15-kDa inner membrane protein subunit of the Tol-Pal complex in Gram-negative bacteria, and its function is poorly understood. Tol-Pal is recruited to cell division sites where it is involved in maintaining the integrity of the outer membrane. TolR is related to MotB, the peptidoglycan (PG)-binding stator protein from the flagellum, suggesting it might serve a similar role in Tol-Pal. The only structure thus far reported for TolR is of the periplasmic domain from Haemophilus influenzae in which N- and C-terminal residues had been deleted (TolR(62–133), Escherichia coli numbering). H. influenzae TolR(62–133) is a symmetrical dimer with a large deep cleft at the dimer interface. Here, we present the 1.7-Å crystal structure of the intact periplasmic domain of E. coli TolR (TolR(36–142)). E. coli TolR(36–142) is also dimeric, but the architecture of the dimer is radically different from that of TolR(62–133) due to the intertwining of its N and C termini. TolR monomers are rotated ∼180° relative to each other as a result of this strand swapping, obliterating the putative PG-binding groove seen in TolR(62–133). We found that removal of the strand-swapped regions (TolR(60–133)) exposes cryptic PG binding activity that is absent in the full-length domain. We conclude that to function as a stator in the Tol-Pal complex dimeric TolR must undergo large scale structural remodeling reminiscent of that proposed for MotB, where the N- and C-terminal sequences unfold in order for the protein to both reach and bind the PG layer ∼90 Å away from the inner membrane.     

Domain Analysis of ArcS,the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System,Reveals Functional Differentiation of Its Two Receiver Domains

In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli.