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 degrees in comparison with the open form. Here we show an alternative 39 degrees-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N-acetyl-D-galactosamine residue (C Delta 4S6S). In C Delta 4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47 degrees-closed conformations. Therefore, the two sulfate groups in C Delta 4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with C Delta 4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of C Delta 4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that C Delta 4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.