myo-inositol and D-ribose ligand discrimination in an ABC periplasmic binding protein

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.

Fig 1

Model of myo-inositol uptake and catabolism in Caulobacter. When present in the environment, myo-inositol is bound by the PBP IbpA, which contacts the transmembrane transporter IatP and delivers the sugar to the cytoplasmic space. Translocation of myo-inositol (red circle) from the periplasm to the cytoplasm is energized by the hydrolysis of ATP by the ABC protein IatA. myo-Inositol in the cytoplasm is then catabolized, and the late pathway intermediate 2-keto-5-deoxy-d-gluconate-6-phosphate (orange circle) functions as the inducer of the iol genes by inhibiting the IolR response regulator through a direct interaction with its sugar-binding domain (SBD). Genes (idhA, iolC, and ibpA) normally repressed in the absence of myo-inositol are transcribed, facilitating the metabolism of myo-inositol. OM, outer membrane; IM, inner membrane.

Fig 2

Structure of the IbpA protein bound to myo-inositol. (A) Ribbon structure of IbpA. α-Helices (light gray) and β-strands (light pink) are numbered. (B) Simulated annealing composite omit map (contoured at 2σ) of bound myo-inositol in the IbpA ligand-binding cavity. (C) Interaction map of the IbpA side chain-ligand interactions in the IbpA/myo-inositol structure. Measured distances from side chain nitrogen and oxygen atoms to the hydroxyl oxygens of myo-inositol are shown in green. myo-Inositol carbons are numbered in panels B and C.

Fig 3

Structural comparison of C. crescentus IbpA and T. tengcongensis RBP. (A) Amino acid sequence alignment of IbpA (upper sequence) and RBP_Tt (lower sequence). Solid and open circles highlight residues involved in polar interactions and hydrophobic interactions, respectively, between IbpA and myo-inositol (blue) and between RBP_Tt and ribose (orange). Red boxes highlight residues N168 and S203 from IbpA and G169 and F203 from RBP_Tt (see Fig. 5). Red lines highlight residues present in the three-segment hinge. α-Helices are represented by cylinders, and β-strands are represented by arrows. The residue at the beginning of each line is numbered. (B) Structural alignment between IbpA (light blue) and RBP_Tt (light orange, PDB code 2IOY). RMSD = 1.03 Å. (C) Structural alignment of the binding cavity residues of IbpA (light blue) and RBP_Tt (light orange). myo-Inositol and d-ribose are in blue and orange, respectively. To improve the visibility of the side chains presented, F51 and F52 in IbpA and F52 and F53 in RBP_Tt are not shown. The carbons of each ligand are been numbered in blue for myo-inositol and in red for d-ribose.

Fig 4

Neighbor-joining phylogenetic tree of IBPs and RBPs. The tree is constructed from six IBP sequences (C. crescentus [Cc], S. meliloti [Sm], M. loti [Ml], A. tumefaciens [At], B. melitensis [Bm], and Pseudomonas sp. strain GM48 [Ps]) and five RBP sequences (E. coli [Ec], S. enterica serovar Typhimurium [St], B. subtilis [Bs], T. tengcongensis [Tt], and T. maritima [Tm]). Xylose-binding protein sequences from E. coli (Ec) and T. ethanolicus (Te) are the outgroup. Bootstrap values for 100 replicates are presented at the nodes on the tree.

Fig 5

Comparative residue frequencies in the IBP and RBP ligand-binding cavities. Residue numbering is based on the IbpA sequence. For the amino acid sequence alignment used to produce this figure, see Fig. S1 in the supplemental material. (A) Residue frequency in the ligand-binding cavities of IBPs. The residue at each of these positions in Caulobacter IbpA is in red. (B) Residue frequency in the ligand-binding cavities of RBPs. The residue at each of these positions in Caulobacter IbpA is in red. The residues at positions 169 and 174 are not involved in ligand interaction and are in gray. (C) Spatial organization of the N168-S203 pair in IbpA and the G169-F203 pair in RBP_Tt (PDB code 2IOY). myo-Inositol and ribose carbons are numbered.

Fig 6

ITC carbohydrate binding assays. In each panel, the raw heat signal is displayed above the integrated and fitted data (below). Equilibrium dissociation constants (K_d values) were calculated for each titration after subtraction of the buffer heat of dilution. (A) Binding interaction between IbpA and myo-inositol. WT, wild type. (B) Binding interaction between IbpA_CM1 and myo-inositol. (C) Binding interaction between IbpA_CM2 and myo-inositol. (D) Binding interaction between IbpA and d-ribose. (E) Binding interaction between IbpA_CM1 and d-ribose. (F) Binding interaction between IbpA_CM2 and d-ribose.

Fig 7

Effects of ABC transporter mutations and IbpA cavity mutations on C. crescentus growth on myo-inositol and d-ribose. (A) Folding of the different IbpA CMs (CM1 and CM2) was compared to that of the wild-type (WT) protein by UV CD spectroscopy. The α-helix and β-strand contents of each protein were calculated from the CD spectra and compared to the IbpA crystal structure. mdeg, millidegrees. (B) Growth curves of wild-type C. crescentus, ABC transporter mutants, and IbpA CMs in M2 defined medium supplemented with 0.2% (wt/vol) myo-inositol. (C) Growth curves of wild-type C. crescentus, ABC transporter mutants, and IbpA CMs in M2 defined medium supplemented with 0.2% (wt/vol) d-ribose.