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

The Journal of Biological Chemistry
Marcus FischerG H Thomas

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 substra...Continue Reading

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Citations

Jan 12, 2017·Biophysical Journal·Janin GlaenzerGregor Hagelueken
Apr 14, 2017·Molecular Microbiology·Gavin H Thomas
Feb 9, 2017·Acta Crystallographica. Section D, Structural Biology·Robert A Nicholls
Jun 11, 2016·Biochemical Society Transactions·Gavin H Thomas
Feb 27, 2018·Frontiers in Cellular and Infection Microbiology·Leonardo T RosaDavid J Kelly
May 3, 2018·Nature Communications·Weixiao Y WahlgrenRosmarie Friemann
Jul 17, 2021·Frontiers in Molecular Biosciences·James S DaviesRenwick C J Dobson
Jul 1, 2021·Environmental Microbiology·Jennifer B GlassFrank J Stewart
Sep 14, 2019·Journal of the American Chemical Society·John F DarbyMarcus Fischer

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Methods Mentioned

BETA
PCR
Fluorescence
protein folding
isothermal titration calorimetry
ion-exchange chromatography

Software Mentioned

SCALA
Comit
Magellan
Procheck
Refmac
Std
MOSFLM
Sfcheck
DataMax
ACORN

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