myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

Abstract

ABSTRACT The periplasmic binding protein (PBP) IbpA mediates the uptake of myo -inositol by the IatP-IatA ATP-binding cassette transmembrane transporter. We report a crystal structure of Caulobacter crescentus IbpA bound to myo -inositol at 1.45 Å resolution. This constitutes the first structure of a PBP bound to inositol. IbpA adopts a type I PBP fold consisting of two α-β lobes that surround a central hinge. A pocket positioned between the lobes contains the myo -inositol ligand, which binds with submicromolar affinity (0.76 ± 0.08 μM). IbpA is homologous to ribose-binding proteins and binds d -ribose with low affinity (50.8 ± 3.4 μM). On the basis of IbpA and ribose-binding protein structures, we have designed variants of IbpA with inverted binding specificity for myo -inositol and d -ribose. Five mutations in the ligand-binding pocket are sufficient to increase the affinity of IbpA for d -ribose by 10-fold while completely abolishing binding to myo -inositol. Replacement of ibpA with these mutant alleles unable to bind myo -inositol abolishes C. crescentus growth in medium containing myo -inositol as the sole carbon source. Neither deletion of ibpA nor replacement of ibpA with the high-affinity ribose binding allele affected C. crescentus growth on d -ribose as a carbon source, providing evidence that the IatP-IatA transporter is specific for myo -inositol. This study outlines the evolutionary relationship between ribose- and inositol-binding proteins and provides insight into the molecular basis upon which these two related, but functionally distinct, classes of periplasmic proteins specifically bind carbohydrate ligands.