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.
Highlights
Haemophilus influenzae requires a substrate-binding protein (SBP)-dependent Tripartite ATP-independent periplasmic (TRAP) transporter to acquire sialic acid
As we learn about the diversity of bacterial physiology through genome sequencing, it is clear that bacterial TRAP transporters form significant components of the encoded repertoire of solute transporter in the genomes of bacteria that live in nutrient-poor environments (7)
TRAP transporters appear to be enriched in these environments as they have high affinity, use Naϩ as a coupling ion for transport, and have specificity for diverse organic acids that appear to be major carbon sources in these niches (28, 29)
Summary
Haemophilus influenzae requires a substrate-binding protein (SBP)-dependent TRAP transporter to acquire sialic acid. A remarkable recent study has added another 29 unique DctP-TRAP SBP structures to the Protein Data Bank, each with an equivalent arginine (18) These data reinforce the key role of this residue in defining the biological function and substrate range of TRAP transporters, but the structural and biochemical consequences of its disruption have not been investigated to date. In this study we determined the role of the arginine/carboxylate interaction using SiaP We demonstrate that it is essential for high affinity binding of substrate and transport under physiological conditions of low substrate concentrations and that its loss can be tolerated at high ligand concentrations. Together these data demonstrate a key role for the Arg-147/carboxylate interaction in TRAP transporter function
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