Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT_So, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT_So represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.     

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.     

Brucin,an antibacterial peptide derived from fruit protein of Fructus Bruceae,Brucea javanica (L.) Merr

A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse‐phase HPLC, and antimicrobial peptides active against Gram‐negative and Gram‐positive bacteria were analysed using SDS‐PAGE and nanoLC‐MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His‐Thr‐Leu‐Cys‐Met‐Asp‐Gly‐Gly‐Ala‐Thr‐Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11‐amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16‐fold and 12·5‐fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l^?1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes.

Significance and Impact of the Study

An antibacterial peptide, named Brucin with specificity for Streptococcus pyogenes, was produced in vitro from dried fruit protein of Brucea javanica (L.) Merr. by pepsin‐catalysed hydrolysis. Its inhibitory activity towards the Gram‐positive bacteria was higher than penicillin G and chloramphenicol. The result suggested that Brucin may be applied for the treatment of the disease caused by Strep. pyogenes^*.     

Probing receptor-translocator interactions in the oligopeptide ABC transporter by fluorescence correlation spectroscopy

The oligopeptide transporter Opp is a five-component ABC uptake system. The extracytoplasmic lipid-anchored substrate-binding protein (or receptor) OppA delivers peptides to an integral membrane complex OppBCDF (or translocator), where, on ATP binding and hydrolysis, translocation across the membrane takes place. OppA and OppBCDF were labeled with fluorescent probes, reconstituted into giant unilamellar vesicles, and the receptor-translocator interactions were investigated by fluorescence correlation spectroscopy. Lateral mobility of OppA was reduced on incorporation of OppBCDF into giant unilamellar vesicles, and decreased even further on the addition of peptide. Fluorescence cross-correlation measurements revealed that OppBCDF distinguished liganded from unliganded OppA, binding only the former. Addition of ATP or its nonhydrolyzable analog AMP-PNP resulted in release of OppA from OppBCDF. In vanadate-trapped “transition state” conditions, OppA also was not bound by OppBCDF. A model is presented in which ATP-binding to OppDF results in donation of peptide to OppBC and simultaneous release of OppA. ATP-hydrolysis would complete the peptide translocation and reset the transporter for another catalytic cycle. Implications in terms of a general transport mechanism for ABC importers and exporters are discussed.     

Nickel transport systems in microorganisms

The transition metal Ni is an essential cofactor for a number of enzymatic reactions in both prokaryotes and eukaryotes. Molecular analyses have revealed the existence of two major types of high-affinity Ni²⁺ transporters in bacteria. The Nik system of Escherichia coli is a member of the ABC transporter family and provides Ni²⁺ ion for the anaerobic biosynthesis of hydrogenases. The periplasmic binding protein of the transporter, NikA, is likely to play a dual role. It acts as the primary binder in the uptake process and is also involved in negative chemotaxis to escape Ni overload. Expression of the nik operon is controlled by the Ni-responsive repressor NikR, which shows functional similarity to the ferric ion uptake regulator Fur. The second type of Ni²⁺ transporter is represented by HoxN of Ralstonia eutropha, the prototype of a novel family of transition metal permeases. Members of this family have been identified in gram-negative and gram-positive bacteria and recently also in a fission yeast. They transport Ni²⁺ with very high affinity, but differ with regard to specificity. Site-directed mutagenesis experiments have identified residues that are essential for transport. Besides these uptake systems, different types of metal export systems, which prevent microorganisms from the toxic effects of Ni²⁺ at elevated intracellular concentrations, have also been described. Received: 14 July / Accepted: 8 October 1999     

Structural and functional characterization of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy‐terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15 µg/ml) to both Gram‐positive and Gram‐negative bacteria, whereas the parent peptide only exhibited Gram‐positive antibacterial activity. The Os peptide was found to be two‐fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane‐mimicking environment, the cysteine‐containing peptide has a higher α‐helical content. Both peptides were found to be non‐toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA

Recent crystal structures of the multidrug ATP‐binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208′) in the intracellular domains of its two half‐transporters, close to the nucleotide‐binding domains (NBDs), which are in close proximity of each other in the outward‐facing state but not in the inward‐facing state. Using intermolecular cysteine crosslinking between E208C and E208C′ in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide‐free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half‐transporters like a nucleotide‐free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The mechanism of nucleotide‐binding domain dimerization in the intact maltose transporter as studied by all‐atom molecular dynamics simulations

The Escherichia coli maltose transporter MalFGK₂‐E belongs to the protein superfamily of ATP‐binding cassette (ABC) transporters. This protein is composed of heterodimeric transmembrane domains (TMDs) MalF and MalG, and the homodimeric nucleotide‐binding domains (NBDs) MalK₂. In addition to the TMDs and NBDs, the periplasmic maltose binding protein MalE captures maltose and shuttle it to the transporter. In this study, we performed all‐atom molecular dynamics (MD) simulations on the maltose transporter and found that both the binding of MalE to the periplasmic side of the TMDs and binding of ATP to the MalK₂ are necessary to facilitate the conformational change from the inward‐facing state to the occluded state, in which MalK₂ is completely dimerized. MalE binding suppressed the fluctuation of the TMDs and MalF periplasmic region (MalF‐P2), and thus prevented the incorrect arrangement of the MalF C‐terminal (TM8) helix. Without MalE binding, the MalF TM8 helix showed a tendency to intrude into the substrate translocation pathway, hindering the closure of the MalK₂. This observation is consistent with previous mutagenesis experimental results on MalF and provides a new point of view regarding the understanding of the substrate translocation mechanism of the maltose transporter.     

Antibacterial potential of hGlyrichin encoded by a human gene

Emerging multidrug‐resistant (MDR) bacteria are an enormous threat to human life because of their resistance to currently available antibiotics. The genes encoding antibacterial peptides have been studied extensively and are excellent candidates for a new generation of antibiotic drugs to fight MDR bacteria. In contrast to traditional antibiotics, antibacterial peptides, which do not cause drug resistance, have an unparalleled advantage. However, because most antibacterial peptides originate in species other than humans, the hetero‐immunological rejection of antibacterial peptides is a key disadvantage that limits their clinical application. In this study, we identify hGlyrichin as a potential human antibacterial polypeptide. The hGlyrichin polypeptide kills a variety of bacteria including the MDR bacteria methicillin‐resistant Staphylococcus aureus, MDR Pseudomonas aeruginosa, and MDR tubercle bacillus. A 19 amino acid peptide (pCM19) at positions 42–60 of hGlyrichin is crucial for its antibacterial activity. The hGlyrichin polypeptide kills bacteria through the destruction of the bacterial membrane. In addition, all peptides that are homologous to hGlyrichin have antibacterial activity and can penetrate the bacterial membrane. Importantly, hGlyrichin does not cause hemolytic side effects in vitro or in vivo. Therefore, based on the virtues of hGlyrichin, i.e., the absence of hetero‐immunological rejection and hemolytic side effects and the unambiguous efficacy of killing pathogenic MDR bacteria, we propose hGlyrichin as a potential human antibacterial polypeptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.     

Mapping of Apidaecin Regions Relevant for Antimicrobial Activity and Bacterial Internalization

Apidaecins are 18–20-residue long proline-rich peptides expressed in insects as part of the innate immune system. They are very active against Gram-negative bacteria, especially Enterobacteriaceae. The C-terminal sequence PRPPHPRL is highly conserved, whereas the N-terminal region is variable. By replacing all 18 residues of apidaecin 1a and apidaecin 1b individually by alanine (Ala-scan), we have shown that single mutations in the C-terminal half of the peptides drastically reduced and mostly abolished the antibacterial activity against Escherichia coli. Conversely, substitutions in the N-terminal eight residues produced no, or only minor effects. The activity loss was correlated to the ability of apidaecin 1b and its mutants to enter Gram-negative bacteria, most likely because they no longer bind to a protein transporter. This assumed binding, however, was not inhibited by truncated apidaecin peptides added at tenfold higher concentrations. Interestingly, the antibacterial activity of full length apidaecin 1b was enhanced about four times by addition of a N-terminally truncated apidaecin peptide [11–18]-apidaecin 1b, as indicated by lower MIC-values against E. coli, although the short 5(6)-carboxyfluorescein-labeled peptide did not enter the bacteria. In contrast, the activity against the Gram-positive bacterium Micrococcus luteus was not located in the C-terminal sequence of apidaecins 1a and b, but depended mostly on the presence of all four basic residues.     

Escherichia coli yjjPB genes encode a succinate transporter important for succinate production

Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.     

Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N-terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions -1 and -2. A dedicated ATP-binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterlum divergens that is secreted via the cell's general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC-transporter-dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.     

Alternating access mechanism in the POT family of oligopeptide transporters

Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton‐dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3‐Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge‐like movement within the C‐terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide‐binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.     

Allosteric transitions of the maltose transporter studied by an elastic network model

The maltose transporter from Escherichia coli is one of the ATP‐binding cassette (ABC) transporters that utilize the energy from ATP hydrolysis to translocate substrates across cellular membranes. Until 2011, three crystal structures have been determined for maltose transporter at different states in the process of transportation. Here, based on these crystal structures, the allosteric pathway from the resting state (inward‐facing) to the catalytic intermediate state (outward‐facing) is studied by applying an adaptive anisotropic network model. The results suggest that the allosteric transitions proceed in a coupled way. The closing of the nucleotide‐binding domains occurs first, and subsequently this conformational change is propagated to the transmembrane domains (TMD) via the EAA and EAS loops, and then to the maltose‐binding protein, which facilitates the translocation of the maltose. It is also found that there exist nonrigid‐body and asymmetric movements in the TMD. The cytoplasmic gate may only play the role of allosteric propagation during the transition from the pretranslocation to outward‐facing states. In addition, the results show that the movment of the helical subdomain towards the RecA‐like subdomain mainly occurs in the earlier stages of the transition. These results can provide some insights into the understanding of the mechanism of ABC transporters. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 758–768, 2014.     

Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA

Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme–substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme–product complex (trapped by ADP-vanadate). 20 Å resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.     

Novel antibacterial peptides induced by probiotics in Hermetia illucens (Diptera: Stratiomyidae) larvae

There is a need to discover new therapeutic substances due to the emergence of deadly infectious diseases and various antibiotic resistance. We focused on the larvae that are utilized as a medical insect for the treatment of skin damage in Europe and America. This study was to investigate the pharmacological activities of novel antibacterial peptides isolated from Hermetia illucens larvae against the Klebsiella pneumoniae and Shigella dysenteriae. The larvae were immunized by probiotics (Lactobacillus casei) for 24 h. The hemolymph from the immunized larvae was fractionated through reverse‐phase chromatography. Peptides were purified using HPLC and the coomassie blue staining, and identified using Nano‐LC‐ESI‐MS/MS system. Antibacterial activities of the peptides were evaluated by turbidometric assay, liquid broth dilution assay, resazurin assay, and agar disk diffusion method. The minimum inhibitory concentrations (MICs) of the peptides were measured as 150 μg/mL through the turbidometric, liquid broth dilution, and resazurin assays. The peptides effectively inhibited their growth/proliferation as well as the survival rate of the tested bacteria. Furthermore, the immunized larvae exhibited overexpression of the peptides compared to non‐immunized larvae. These results demonstrate that the peptides induced by H. illucens exert strong antibacterial activity against Gram‐negative bacteria. The results suggest that the activation of the humoral immunity induced by immunization functioned to enhance the production of antibacterial peptides from the insect and their antibacterial properties. This study indicates the potential of the peptides produced from larvae as antibacterial peptide substance for the development of novel antibacterial drugs.     

Tuning the Cellular Trafficking of the Lysosomal Peptide Transporter TAPL by its N‐terminal Domain

The homodimeric ATP‐binding cassette (ABC) transport complex TAPL (transporter associated with antigen processing‐like, ABCB9) translocates a broad spectrum of peptides from the cytosol into the lumen of lysosomes. The presence of an extra N‐terminal transmembrane domain (TMD0) lacking any sequence homology to known proteins distinguishes TAPL from most other ABC transporters of its subfamily. By dissecting TAPL, we could assign distinct functions to the core complex and TMD0. The core‐TAPL complex, composed of six predicted transmembrane helices and a nucleotide‐binding domain, is sufficient for peptide transport, showing that the core transport complex is correctly targeted to and assembled in the membrane. Strikingly, in contrast to the full‐length transporter, the core translocation complex is targeted preferentially to the plasma membrane. However, TMD0 alone, comprising a putative four transmembrane helix bundle, traffics to lysosomes. Upon coexpression, TMD0 forms a stable non‐covalently linked complex with the core translocation machinery and guides core‐TAPL into lysosomal compartments. Therefore, TMD0 represents a unique domain, which folds independently and encodes the information for lysosomal targeting. These outcomes are discussed in respect of trafficking, folding and function of TAPL.