scholarly journals OpuF, a NewBacillusCompatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Laura Teichmann ◽  
Henriette Kümmel ◽  
Bianca Warmbold ◽  
Erhard Bremer
Keyword(s):  
Abc Transporters ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Content Type ◽  
High Osmolarity ◽  
Physiological Characterization ◽  
Substrate Binding Protein

ABSTRACTThe accumulation of compatible solutes is a common defense of bacteria against the detrimental effects of high osmolarity. Uptake systems for these compounds are cornerstones in cellular osmostress responses because they allow the energy-preserving scavenging of osmostress protectants from environmental sources.Bacillus subtilisis well studied with respect to the import of compatible solutes and its five transport systems (OpuA, OpuB, OpuC, OpuD, and OpuE), for these stress protectants have previously been comprehensively studied. Building on this knowledge and taking advantage of the unabated appearance of new genome sequences of members of the genusBacillus, we report here the discovery, physiological characterization, and phylogenomics of a new member of the Opu family of transporters, OpuF (OpuFA-OpuFB). OpuF is not present inB. subtilisbut it is widely distributed in members of the large genusBacillus. OpuF is a representative of a subgroup of ATP-binding cassette (ABC) transporters in which the substrate-binding protein (SBP) is fused to the transmembrane domain (TMD). We studied the salient features of the OpuF transporters fromBacillus infantisandBacillus panaciterraeby functional reconstitution in aB. subtilischassis strain lacking known Opu transporters. A common property of the examined OpuF systems is their substrate profile; OpuF mediates the import of glycine betaine, proline betaine, homobetaine, and the marine osmolyte dimethylsulfoniopropionate (DMSP). Anin silicomodel of the SBP domain of the TMD-SBP hybrid protein OpuFB was established. It revealed the presence of an aromatic cage, a structural feature commonly present in ligand-binding sites of compatible solute importers.IMPORTANCEThe high-affinity import of compatible solutes from environmental sources is an important aspect of the cellular defense of many bacteria and archaea against the harmful effects of high external osmolarity. The accumulation of these osmostress protectants counteracts high-osmolarity-instigated water efflux, a drop in turgor to nonphysiological values, and an undue increase in molecular crowding of the cytoplasm; they thereby foster microbial growth under osmotically unfavorable conditions. Importers for compatible solutes allow the energy-preserving scavenging of osmoprotective and physiologically compliant organic solutes from environmental sources. We report here the discovery, exemplary physiological characterization, and phylogenomics of a new compatible solute importer, OpuF, widely found in members of theBacillusgenus. The OpuF system is a representative of a growing subgroup of ABC transporters in which the substrate-scavenging function of the substrate-binding protein (SBP) and the membrane-embedded substrate translocating subunit (TMD) are fused into a single polypeptide chain.

scholarly journals Identification and Characterization of an ATP Binding Cassette l-Carnitine Transporter inListeria monocytogenes

2000 ◽  
Vol 66 (11) ◽  
pp. 4696-4704 ◽  
Author(s):  
Katy R. Fraser ◽  
Duncan Harvie ◽  
Peter J. Coote ◽  
Conor P. O'Byrne
Keyword(s):  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Brain Heart Infusion ◽  
Defined Medium ◽  
Compatible Solute ◽  
Atp Binding ◽  
Insertional Inactivation ◽  
Substrate Binding Protein ◽  
Atp Binding Cassette

ABSTRACT We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of theopuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake ofl-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting l-carnitine and which plays an important role in osmoregulation in this pathogen.

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC

2011 ◽  
Vol 436 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Yang Du ◽  
Wei-Wei Shi ◽  
Yong-Xing He ◽  
Yi-Hu Yang ◽  
Cong-Zhao Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Structural Basis ◽  
Binding Property ◽  
Substrate Binding Protein

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes the multiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr94 in OpuCC compared with Asp96 in OpuBC.

scholarly journals The Compatible-Solute-Binding Protein OpuAC from Bacillus subtilis: Ligand Binding, Site-Directed Mutagenesis, and Crystallographic Studies

2008 ◽  
Vol 190 (16) ◽  
pp. 5663-5671 ◽  
Author(s):  
Sander H. J. Smits ◽  
Marina Höing ◽  
Justin Lecher ◽  
Mohamed Jebbar ◽  
Lutz Schmitt ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Site Directed Mutagenesis ◽  
Transport Systems ◽  
The Past ◽  
Substrate Binding Protein

ABSTRACT In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-Å resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.

scholarly journals Molecular Basis of Unexpected Specificity of ABC Transporter-Associated Substrate-Binding Protein DppA from Helicobacter pylori

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Mohammad M. Rahman ◽  
Mayra A. Machuca ◽  
Mohammad F. Khan ◽  
Christopher K. Barlow ◽  
Ralf B. Schittenhelm ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Content Type ◽  
H Pylori ◽  
Substrate Binding Protein ◽  
Broad Specificity

ABSTRACT The gastric pathogen Helicobacter pylori has limited ability to use carbohydrates as a carbon source, relying instead on exogenous amino acids and peptides. Uptake of certain peptides by H. pylori requires an ATP binding cassette (ABC) transporter annotated dipeptide permease (Dpp). The transporter specificity is determined by its cognate substrate-binding protein DppA, which captures ligands in the periplasm and delivers them to the permease. Here, we show that, unlike previously characterized DppA proteins, H. pylori DppA binds, with micromolar affinity, peptides of diverse amino acid sequences ranging between two and eight residues in length. We present analysis of the 1.45-Å-resolution crystal structure of its complex with the tetrapeptide STSA, which provides a structural rationale for the observed broad specificity. Analysis of the molecular surface revealed a ligand-binding pocket that is large enough to accommodate peptides of up to nine residues in length. The structure suggests that H. pylori DppA is able to recognize a wide range of peptide sequences by forming interactions primarily with the peptide main chain atoms. The loop that terminates the peptide-binding pocket in DppAs from other bacteria is significantly shorter in the H. pylori protein, providing an explanation for its ability to bind longer peptides. The subsites accommodating the two N-terminal residues of the peptide ligand make the greatest contribution to the protein-ligand binding energy, in agreement with the observation that dipeptides bind with affinity close to that of longer peptides. IMPORTANCE The World Health Organization listed Helicobacter pylori as a high-priority pathogen for antibiotic development. The potential of using peptide transporters in drug design is well recognized. We discovered that the substrate-binding protein of the ABC transporter for peptides, termed dipeptide permease, is an unusual member of its family in that it directly binds peptides of diverse amino acid sequences, ranging between two and eight residues in length. We also provided a structural rationale for the observed broad specificity. Since the ability to import peptides as a source of carbon is critical for H. pylori, our findings will inform drug design strategies based on inhibition or fusion of membrane-impermeant antimicrobials with peptides.

scholarly journals Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Julia Cuellar ◽  
Mia Åstrand ◽  
Heli Elovaara ◽  
Annukka Pietikäinen ◽  
Saija Sirén ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Cellular Localization ◽  
De Novo ◽  
Binding Protein ◽  
Substrate Binding ◽  
Nucleoside Transporter ◽  
Bacterial Survival ◽  
Content Type ◽  
Transporter Family ◽  
Substrate Binding Protein

ABSTRACT Borrelia burgdorferi sensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato. Because B. burgdorferi sensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

scholarly journals Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis

2014 ◽  
Vol 82 (8) ◽  
pp. 3503-3512 ◽  
Author(s):  
Taketo Otsuka ◽  
Charmaine Kirkham ◽  
Antoinette Johnson ◽  
Megan M. Jones ◽  
Timothy F. Murphy
Keyword(s):  
Otitis Media ◽  
Moraxella Catarrhalis ◽  
Binding Protein ◽  
Substrate Binding ◽  
Vaccine Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Chronic Obstructive ◽  
Content Type ◽  
Substrate Binding Protein

ABSTRACTMoraxella catarrhalisis a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeableHaemophilus influenzae,M. catarrhalishas become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, ofM. catarrhalis. Among 30 clinical isolates tested, thesbp2gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not thesbp2mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance ofM. catarrhalisfrom the lung compared to that in the control group at both 25-μg and 50-μg doses (P< 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen againstM. catarrhalis.

scholarly journals The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis

2015 ◽  
Vol 84 (2) ◽  
pp. 432-438 ◽  
Author(s):  
Taketo Otsuka ◽  
Charmaine Kirkham ◽  
Aimee Brauer ◽  
Mary Koszelak-Rosenblum ◽  
Michael G. Malkowski ◽  
...  
Keyword(s):  
Amino Acids ◽  
Respiratory Tract ◽  
Abc Transporter ◽  
Moraxella Catarrhalis ◽  
Vaccine Candidate ◽  
Binding Protein ◽  
Substrate Binding ◽  
Content Type ◽  
Basic Amino Acids ◽  
Substrate Binding Protein

Moraxella catarrhalisis an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to preventM. catarrhalisinfections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface ofM. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement ofM. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth ofM. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen.

The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity

2015 ◽  
Vol 43 (5) ◽  
pp. 1011-1017 ◽  
Author(s):  
Abbas Maqbool ◽  
Richard S.P. Horler ◽  
Axel Muller ◽  
Anthony J. Wilkinson ◽  
Keith S. Wilson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Abc Transporters ◽  
Binding Protein ◽  
Substrate Binding ◽  
Structure And Function ◽  
High Affinity ◽  
A Subunit ◽  
And Function ◽  
Substrate Binding Protein

ATP-binding cassette (ABC) transporters, although being ubiquitous in biology, often feature a subunit that is limited primarily to bacteria and archaea. This subunit, the substrate-binding protein (SBP), is a key determinant of the substrate specificity and high affinity of ABC uptake systems in these organisms. Most prokaryotes have many SBP-dependent ABC transporters that recognize a broad range of ligands from metal ions to amino acids, sugars and peptides. Herein, we review the structure and function of a number of more unusual SBPs, including an ABC transporter involved in the transport of rare furanose forms of sugars and an SBP that has evolved to specifically recognize the bacterial cell wall-derived murein tripeptide (Mtp). Both these examples illustrate that subtle changes in binding-site architecture, including changes in side chains not directly involved in ligand co-ordination, can result in significant alteration of substrate range in novel and unpredictable ways.

scholarly journals Characterization and protective activity of monoclonal antibodies directed against Fe (3+) ABC transporter substrate-binding protein of Glaesserella parasuis

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Kexin Zhu ◽  
Dong Yu ◽  
Jiahui An ◽  
Yufeng Li
Keyword(s):  
Monoclonal Antibodies ◽  
Abc Transporter ◽  
Subunit Vaccine ◽  
Binding Protein ◽  
Substrate Binding ◽  
Passive Immunization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
And Control ◽  
Substrate Binding Protein

AbstractGlässer’s disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p < 0.01) in comparison with the control group. Opsonophagocytic assays demonstrated that mAb 2D1 significantly enhanced the phagocytosis of 3D4/21 cells by G. parasuis (p < 0.05). RAW264.7 cells with stronger phagocytic ability were also used for the opsonophagocytic assay, and the difference was highly significant (p < 0.01). Passive immunization of mice revealed that mAb 2D1 can eliminate the bacteria in the blood and provide protection against G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.

Sign in / Sign up

Export Citation Format

Share Document