Alternative substrate-bound conformation of bacterial solute-binding protein involved in the import of mammalian host glycosaminoglycans

Abstract

Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis, the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by polysaccharide lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N-acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.

Figure 1

Gram-negative Streptobacillus GAG import system. (A) A model for GAG (chondroitin sulfate C) import. Polysaccharide GAGs are depolymerized to unsaturated disaccharides by extracellular or cell-surface polysaccharide lyases. Unsaturated GAG disaccharides are incorporated in the cytoplasm by the ABC transporter (Smon0121-Smon0122/Smon0120-Smon0120) through the periplasmic solute-binding protein (Smon0123). Incorporated disaccharides are degraded to monosaccharides by cytoplasmic UGL and are metabolized by some enzymes. (B) Structural formulas of unsaturated chondroitin disaccharides. (C) The degradation of chondroitin sulfate A. Upper, before adding acetic acid; and lower, after adding acetic acid. Left, S. moniliformis; middle, Pedobacter heparinus as the positive control; and right, Escherichia coli as the negative control. (D) Decrease in intensity of Smon0123 by adding increasing CΔ4S6S was plotted after a modification based on the volume change in the cuvette.

Figure 2

Three-dimensional structure of CΔ4S6S-bound Smon0123. Overall structure (A) and the binding mode of Smon0123 to CΔ4S6S (B). (A) Green, N1 subdomain; light green, N2 subdomain; orange, C1 subdomain; and light orange, C2 subdomain. The ball model shows CΔ4S6S (pink, carbon atom; red, oxygen atom; blue, nitrogen atom; and yellow, sulfur atom). Gray ball shows the calcium ion. (B) The pink stick model shows CΔ4S6S (pink, carbon atom; red, oxygen atom; blue, nitrogen atom; and yellow, sulfur atom). Stick models show Smon0123 amino acid residues that interact with CΔ4S6S via hydrogen bonds and/or van der Waals contacts (green/light green/orange, carbon atom; red, oxygen atom; and blue, nitrogen atom). Dark red and cyan dashed lines show direct and indirect hydrogen bonds between Smon0123 and CΔ4S6S, respectively. Small cyan balls show water molecules. (C) Electrostatic potential of the substrate-binding pocket. The stick model shows CΔ4S6S. Blue and red surfaces refer to positively and negatively charged spaces, respectively.

Figure 3

The torsion angle differences of substrate-free and substrate-bound Smon0123. Differences of Cα-torsion angles between substrate-free and CΔ0S-bound Smon0123 (A), CΔ4S-bound Smon0123 (B), CΔ6S-bound Smon0123 (C), and CΔ4S6S-bound Smon0123 (D). Black, φ plot; gray, ψ plot. Arrow shows the peaks around Gly265 and Ser470.

Figure 4

Interdomain interactions by substrate binding. Interdomain differences between substrate-free (left) and substrate-bound (right) Smon0123. Green, N1 subdomain; light green, N2 subdomain; orange, C1 subdomain; and light orange, C2 subdomain. (A) Upper, CΔ4S6S-bound Smon0123; and lower, CΔ6S-bound Smon0123. (B) Substrate binding (red circle) induces the changes of hydrogen bonds between the subdomains. In substrate-bound Smon0123, hydrogen bonds were formed between N1 and C2 subdomains (red broken line), which disappeared and were alternatively formed between N2 and C1 subdomains (red line) compared with substrate-free Smon0123.

Figure 5

Hinge-bending motion of substrate-free and substrate-bound Smon0123. (A) N domains of substrate-free and substrate-bound Smon0123 were superimposed. Gray, CΔ0S-bound; green, CΔ4S-bound; blue, CΔ6S-bound; pink, CΔ4S6S-bound; and olive, substrate-free Smon0123. Ribbon models and ball models show the main chain of Smon0123 and disaccharides, respectively. Although the domains of CΔ0S-, CΔ4S-, and CΔ6S-bound Smon0123 were 47° more closed than those of substrate-free Smon0123 (left), the hinge-bending motion of CΔ4S6S-bound Smon0123 was 39° (right). (B) Smon0123 amino acid residues directly formed hydrogen bonds with CΔ0S (gray), CΔ4S (green), CΔ6S (blue), and CΔ4S6S (pink). (C) Some residues of CΔ4S6S-bound Smon0123 (pink) located in a different position from those in the other substrate-bound Smon0123 (gray, CΔ0S; green, CΔ4S; and blue, CΔ6S). The stick models show unsaturated chondroitin disaccharides, and the line models show Arg204, Lys210, Trp284, and Ser287 residues. Small cyan balls show water molecules (oxygen atoms). Magenta and cyan dashed lines show direct and indirect hydrogen bonds, respectively.

Figure 6

Interaction between Smon0123 and Smon0121-Smon-0122/Smon0120-Smon0120. Structure modeling of the complex of CΔ6S-bound Smon0123 (A) or CΔ4S6S-bound Smon0123 (B) and Smon0121-Smon0122/Smon0120-Smon0120. Dark blue, Smon0121; light green, Smon0122; cyan and light orange, Smon0120; blue, Smon0123/CΔ6S; and pink, Smon0123/CΔ4S6S. The olive ball shows calcium ion. (B) The superimposition of CΔ6S-bound Smon0123 (C) or CΔ4S6S-bound Smon0123 (D) on AlgQ2 (gray). (E) ATPase activity of the Smon0121-Smon0122/Smon0120-Smon0120 in liposomes in the absence of disaccharide (PLS, proteoliposome) or presence of CΔ0S, CΔ4S, CΔ6S, CΔ4S6S, and cellobiose. Each data represents the average of triplicate individual experiments (means ± standard errors of the means). Student’s t-test, P > 0.05.