Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding

Fischer, Marcus; Hopkins, Adam; Severi, Emmanuele; Hawkhead, Judith A.; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

doi:10.1074/jbc.m115.656603

Cited by 41 publications

(48 citation statements)

References 37 publications

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“…Further structures might reveal whether this is a feature common to the whole family, as suggested by sequence alignments [11]. In addition, the solvation provided by the presence of water molecules may dissipate the charge, as shown previously for TRAP transporters [35]. This repulsive negative charge might, however, be compensated somewhat by the dipoles of helices a1 and a4 and possibly to some extent a5 which are directed towards the carboxylate groups of the substrate.…”

Section: Discussionmentioning

confidence: 68%

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

et al. 2017

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Section: Discussionmentioning

confidence: 68%

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

et al. 2017

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“…Available crystal structures of SBPs indicate that these proteins have individually defined binding sites in which the ligands are coordinated by hydrogen bonds, hydrophobic interactions and one single salt bridge mediated by a highly conserved arginine (M€ uller et al, 2006;Lecher et al, 2009). Recently, Fischer and colleagues showed that the interaction of arginine and the carboxylate restricts the substrate range to carboxylate containing ligands (Fischer et al, 2015). In DctP Am , this arginine residue is located at position 175 in DctP Am and most probably forms a salt bridge with the carboxylic group of D-gluconic acid, D-fuconic acid, D-xylonic acid or D-galactonic acid, respectively.…”

Section: Discussionmentioning

confidence: 99%

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7^T is mediated by periplasmic sugar oxidation and a TRAP‐transport system

Meinert

Senger

Witthohn

et al. 2017

Molecular Microbiology

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In this study, we investigated an SBP (DctP ) of a tripartite ATP-independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7 . Deletion of dctP as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7 , if cultivated on mineral salt medium supplemented with d-glucose, d-galactose, l-arabinose, d-fucose, d-xylose or d-gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctP using the broad host vector pBBR1MCS-5::dctP . Furthermore, an uptake assay with radiolabeled [ C(U)]-d-glucose clearly showed that the deletion of dctP , dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of K performing thermal shift assays showed a shift in the melting temperature of DctP in the presence of d-gluconic acid (K 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above-mentioned carbohydrates d-galactonate (K 10.72 ± 1.4 µM), d-fuconic acid (K 13.50 ± 1.6 µM) and d-xylonic acid (K 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.

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“…Molecules known to be transported by TRAP transporters range from small organic acids including C4-dicarboxylates, larger sugar acids like N-acetylneuraminic acid (Neu5Ac), to amino acids (4,8). Most TRAP transporter substrates contain a carboxylic acid group, which is specifically recognized by the P domain of the transporter (9).…”

Section: Introductionmentioning

confidence: 99%

“…This residue is crucial for the substrate interaction by recognizing the aforementioned carboxylic acid group in the substrate. It thereby acts as a selectivity filter for the transporter, allowing the SBP to recognize organic acids with high affinity and specificity (9). Thus, P domains are structurally well characterized in their two resting states, namely ''open ligand-free'' and ''closed ligand-bound'', and the interactions between substrate and protein are well studied.…”

Section: Introductionmentioning

confidence: 99%

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an openunliganded to closed-liganded state during the transport cycle.

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Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding

Cited by 41 publications

References 37 publications

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7^T is mediated by periplasmic sugar oxidation and a TRAP‐transport system

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

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Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding

Cited by 41 publications

References 37 publications

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

Structural basis for high‐affinity adipate binding to AdpC (RPA4515), an orphan periplasmic‐binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP‐transport system

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

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Carbohydrate uptake in Advenella mimigardefordensis strain DPN7^T is mediated by periplasmic sugar oxidation and a TRAP‐transport system