Abstract
The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell. The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host-derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae SiaPs bind the sialic acid N-acetylneuraminic acid (Neu5Ac) with nanomolar affinity by forming electrostatic and hydrogen-bonding interactions. Here, we report the crystal structure of a periplasmic binding protein (SatA) of the ATP-binding cassette (ABC) transport system from the pathogenic bacterium Haemophilus ducreyi The structure of Hd-SatA in the native form and sialic acid-bound forms (with Neu5Ac and N-glycolylneuraminic acid (Neu5Gc)), determined to 2.2, 1.5, and 2.5 Å resolutions, respectively, revealed a ligand-binding site that is very different from those of the SiaPs of the TRAP transport system. A structural comparison along with thermodynamic studies suggested that similar affinities are achieved in the two classes of proteins through distinct mechanisms, one enthalpically driven and the other entropically driven. In summary, our structural and thermodynamic characterization of Hd-SatA reveals that it binds sialic acids with nanomolar affinity and that this binding is an entropically driven process. This information is important for future structure-based drug design against this pathogen and related bacteria.
Highlights
The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell
The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host– derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae
Sialic acids are transported across the bacterial periplasmic membrane by tripartite ATP-independent periplasmic (TRAP), ATP-binding cassette (ABC), or major facilitator superfamily (MFS) transport systems
Summary
Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; SatA, sialic acid transport A gene; Hd-SatA, H. ducreyi sialic acid transport A gene; SiaP, sialic acid– binding protein; TRAP, tripartite ATP-independent periplasmic; ITC, isothermal calorimetry; LOS, lipooligosaccharide; PDB, Protein Data Bank; MBP, maltose-binding protein. Haemophilus ducreyi, another Gram-negative bacteria, has been reported to use the ABC transport system for sialic acid transport, utilizing ATP hydrolysis to import sugars across the periplasmic membrane [23, 24]. Similar to the maltose-binding protein system, it is expected that SatA bound to Neu5Ac/Neu5Gc anchors onto the transmembrane domain of the ABC transporter. This signals ATP binding, the sialic acid is released from SatA, and the sugar is transported from the periplasm to the cytoplasm by SatBCD [37,38,39]. We report the structures of the sialic acid– binding protein of H. ducreyi, which uses the ABC transporter system (HdSatA), in unliganded and ligand-bound forms. Site-directed mutagenesis of residues that bind to the sugars followed by measurement of binding affinities shed light on the contribution of these residues to the binding phenomenon
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