scholarly journals The structure of HiSiaQM defines the architecture of tripartite ATP-independent periplasmic (TRAP) transporters

2021 ◽  
Author(s):  
Martin F. Peter ◽  
Peer Depping ◽  
Niels Schneberger ◽  
Emmanuele Severi ◽  
Karl Gatterdam ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Pathogenic Bacteria ◽  
Binding Protein ◽  
Substrate Binding ◽  
Acid Uptake ◽  
Transmembrane Helices ◽  
Secondary Transporter ◽  
Lipid Nanodiscs ◽  
Substrate Binding Protein

SummaryTripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria and archaea and provide important uptake routes for many metabolites 1–3. They consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains) that form a functional unit 4. While the structures of the P-domains are well-known, an experimental structure of any QM-domain has been elusive. HiSiaPQM is a TRAP transporter for the monocarboxylate sialic acid, which plays a key role in the virulence of pathogenic bacteria 5. Here, we present the first cryo-electron microscopy structure of the membrane domains of HiSiaPQM reconstituted in lipid nanodiscs. The reconstruction reveals that TRAP transporters consist of 15 transmembrane helices and are structurally related to elevator-type transporters, such as GltPh and VcINDY 6, 7. Whereas the latter proteins function as multimers, the idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. Structural and mutational analyses together with an AlphaFold 8 model of the tripartite (PQM) complex reveal the structural and conformational coupling of the substrate-binding protein to the transporter domains. Furthermore, we characterize high-affinity VHHs that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake in vivo. Thereby, they also confirm the orientation of the protein in the membrane. Our study provides the first structure of any binding-protein dependent secondary transporter and provides starting points for the development of specific inhibitors.

scholarly journals Triggering Closure of a Sialic Acid TRAP Transporter Substrate Binding Protein through Binding of Natural or Artificial Substrates

2021 ◽  
Vol 433 (3) ◽  
pp. 166756
Author(s):  
Martin F. Peter ◽  
Christian Gebhardt ◽  
Janin Glaenzer ◽  
Niels Schneberger ◽  
Marijn de Boer ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Binding Protein ◽  
Substrate Binding ◽  
Artificial Substrates ◽  
Substrate Binding Protein

scholarly journals Triggering closure of a sialic acid TRAP transporter substrate binding protein through binding of natural or artificial substrates

2020 ◽  
Author(s):  
Martin F. Peter ◽  
Christian Gebhardt ◽  
Janin Glaenzer ◽  
Niels Schneberger ◽  
Marijn de Boer ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Single Molecule ◽  
Binding Protein ◽  
Substrate Binding ◽  
Artificial Substrates ◽  
Closed State ◽  
Single Molecule Fret ◽  
Natural Ligand ◽  
Starting Point ◽  
Substrate Binding Protein

AbstractThe pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.

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.

scholarly journals Substrate recognition and ATPase activity of the E. coli cysteine/cystine ABC transporter YecSC-FliY

2020 ◽  
Vol 295 (16) ◽  
pp. 5245-5256 ◽  
Author(s):  
Siwar Sabrialabed ◽  
Janet G. Yang ◽  
Elon Yariv ◽  
Nir Ben-Tal ◽  
Oded Lewinson
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Binding Protein ◽  
Substrate Binding ◽  
Transporter Family ◽  
Wide Range ◽  
Substrate Binding Protein ◽  
Sulfur Containing ◽  
Sulfur Containing Compounds

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

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 Basis of Glycerophosphodiester Recognition by the Mycobacterium tuberculosis Substrate-Binding Protein UgpB

2019 ◽  
Vol 14 (9) ◽  
pp. 1879-1887 ◽  
Author(s):  
Jonathan S. Fenn ◽  
Ridvan Nepravishta ◽  
Collette S. Guy ◽  
James Harrison ◽  
Jesus Angulo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Protein ◽  
Substrate Binding ◽  
Structural Basis ◽  
Substrate Binding Protein

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.

scholarly journals The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter

2009 ◽  
Vol 106 (6) ◽  
pp. 1778-1783 ◽  
Author(s):  
Christopher Mulligan ◽  
Eric R. Geertsma ◽  
Emmanuele Severi ◽  
David J. Kelly ◽  
Bert Poolman ◽  
...  
Keyword(s):  
Binding Protein ◽  
Substrate Binding ◽  
Sodium Gradient ◽  
Substrate Binding Protein

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.

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