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Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.
J Biol Chem 2015; 290(45):27113-23JB

Abstract

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.

Authors+Show Affiliations

From the York Structural Biology Laboratory, Departments of Chemistry and.Biology (Area 10), University of York, P. O. Box 373, York YO10 5YW and.From the York Structural Biology Laboratory, Departments of Chemistry and.Biology (Area 10), University of York, P. O. Box 373, York YO10 5YW and.Biology (Area 10), University of York, P. O. Box 373, York YO10 5YW and.the Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.From the York Structural Biology Laboratory, Departments of Chemistry and.Biology (Area 10), University of York, P. O. Box 373, York YO10 5YW and gavin.thomas@york.ac.uk.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26342690

Citation

Fischer, Marcus, et al. "Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding." The Journal of Biological Chemistry, vol. 290, no. 45, 2015, pp. 27113-23.
Fischer M, Hopkins AP, Severi E, et al. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding. J Biol Chem. 2015;290(45):27113-23.
Fischer, M., Hopkins, A. P., Severi, E., Hawkhead, J., Bawdon, D., Watts, A. G., ... Thomas, G. H. (2015). Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding. The Journal of Biological Chemistry, 290(45), pp. 27113-23. doi:10.1074/jbc.M115.656603.
Fischer M, et al. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding. J Biol Chem. 2015 Nov 6;290(45):27113-23. PubMed PMID: 26342690.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding. AU - Fischer,Marcus, AU - Hopkins,Adam P, AU - Severi,Emmanuele, AU - Hawkhead,Judith, AU - Bawdon,Daniel, AU - Watts,Andrew G, AU - Hubbard,Roderick E, AU - Thomas,Gavin H, Y1 - 2015/09/05/ PY - 2015/04/03/received PY - 2015/9/7/entrez PY - 2015/9/8/pubmed PY - 2016/5/7/medline KW - bacterial pathogenesis KW - membrane transporter reconstitution KW - protein structure KW - sialic acid KW - transporter SP - 27113 EP - 23 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 290 IS - 45 N2 - Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/26342690/Tripartite_ATP_independent_Periplasmic__TRAP__Transporters_Use_an_Arginine_mediated_Selectivity_Filter_for_High_Affinity_Substrate_Binding_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=26342690 DB - PRIME DP - Unbound Medicine ER -