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

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.

enzymes, i.e. isomerase, NADH-dependent reductase, kinase, and aldolase, subsequently metabolize the unsaturated uronate to pyruvate and glyceraldehyde-3-phosphate 7 . The genes encoding these enzymes that are essential for depolymerization, degradation, and metabolism of GAGs assemble a cluster in the bacterial genome. We have recently identified a solute-binding protein-dependent ATP-binding cassette (ABC) transporter to be the first bacterial import system of both sulfated and non-sulfated GAG disaccharides in pathogenic Streptobacillus moniliformis 8 . S. moniliformis, a causative organism of rat bite fever, is characterized by relapsing fever, rash, and migratory polyarthralgias 9 . The solute-binding protein (Smon0123) captures GAG disaccharides in the periplasm and delivers them to the ABC transporter, followed by their import in the cytoplasm (Fig. 1A). The ABC transporter comprises membrane-spanning proteins (Smon0121-Smon0122) as heterodimers and ATPase domains (Smon0120-Smon0120) as homodimers. The solute-binding protein-dependent ABC transporter is also encoded in the GAG genetic cluster. On the basis of the primary structure, the S. moniliformis ABC transporter system ( is similar to the bacterial alginate ABC transporter system (AlgQ2/AlgM1-AlgM2/AlgS-AlgS), the structure of which has been determined in our previous paper 10 .
Chondroitin sulfate comprises d-glucuronic acid (GlcUA)/l-iduronic acid (IdoUA) and N-acetyl-d-galactosamine (GalNAc) 11 , which are linked by a 1,3-glycoside bond. Depending on the position and/or level of the sulfate groups, chondroitin sulfate is divided to several groups such as chondroitin sulfates A and B with a sulfate group at the C-4 position of GalNAc (chondroitin sulfates A and B contain GlcUA and IdoUA, respectively); chondroitin sulfate C with a sulfate group at the C-6 position of GalNAc; chondroitin sulfate D with two sulfate groups at the C-2 position of GlcUA and the C-6 position of GalNAc; and chondroitin sulfate E sulfated at the C-4 and C-6 positions of GalNAc 12 . Unsaturated chondroitin disaccharides are commonly termed as follows: CΔ0S, non-sulfated; CΔ4S, sulfated at the C-4 position of GalNAc; CΔ6S, sulfated at the C-6 position of GalNAc; and CΔ4S6S, sulfated at the C-4 and C-6 positions of GalNAc 13 (Fig. 1B).
ABC transporters in Gram-negative and Gram-positive bacteria generally receive substrates from solute-binding proteins and incorporate them in the cytoplasm [14][15][16] . Because solute-binding proteins have a high affinity with specific substrates [The dissociation constant (K d value) = 0.01-10 μM], these binding proteins play important roles in determining a strict substrate specificity for import 17  These findings provide structural and functional insights for further understanding substrate recognition and import and for developing inhibitors for pathogenic bacteria.

Degradation of chondroitin sulfate A by S. moniliformis. Degradation of chondroitin sulfate A by S.
moniliformis DSM 12112 was investigated using a simple plate method 18 . Bacterial cells were cultured on a plate that contained bovine serum albumin (BSA) and chondroitin sulfate A. The non-degraded GAGs aggregated with BSA to form white precipitates in the presence of acetic acid, whereas degraded GAGs showed clear zoned halos. This simple method is feasible for detecting GAG degradation by various bacteria. In the plate, clear halo zones were observed around S. moniliformis cells, indicating that this bacterium could degrade chondroitin sulfate A (Fig. 1C), as well as hyaluronan and chondroitin sulfate C 8 . We have previously demonstrated that the ABC transporter incorporated GAG disaccharides derived from hyaluronan (ΔHA, unsaturated hyaluronan disaccharide) and chondroitin sulfate (CΔ0S, CΔ4S, and CΔ6S) 8 . This substrate specificity of the ABC transporter suggests that S. moniliformis degrades other chondroitin sulfates that have two sulfate groups in their constitutional units, such as chondroitin sulfates D and E. The plate assay using abundant GAGs was not attempted because chondroitin sulfates D and E are expensive.

Affinity of Smon0123 with chondroitin disaccharides having two sulfate groups.
To investigate the interaction between Smon0123 and CΔ4S6S, the fluorescence intensity of Smon0123, which was derived from tryptophan residues, was measured in the presence of CΔ4S6S (Fig. 1D). The K d value was determined from the plot of the ratio of change in fluorescence intensity, which exhibits a decrease with an increasing CΔ4S6S concentration. The K d value of Smon0123 for CΔ4S6S was determined to be 3.86 ± 0.243 μM, indicating lower affinity than that for other substrates (CΔ0S, 0.635 ± 0.122 μM; CΔ4S, 1.6 ± 0.231 μM; and CΔ6S, 2.76 ± 0.195 μM) 8 . Together with previous results, the binding ability of Smon0123 with CΔ4S6S indicates that the binding protein prefers non-sulfated substrates to sulfated substrates.

Structural determination of CΔ4S6S-bound
Smon0123. X-ray crystallography enables the directly demonstration of the binding mode of Smon0123 with CΔ4S6S. Because the crystals of Smon0123 with the full-length protein gave low-resolution X-ray diffraction data 8 , the N-terminal 18 and C-terminal 5 residues-truncated Smon0123 (N-18/C-5) was used for crystallization. The purified Smon0123 (N-18/C-5) was crystallized in the presence of CΔ4S6S, and the resultant crystal was suitable for determining the structure. The crystal of Smon0123 (N-18/C-5)/CΔ4S6S belongs to a space group P1 and has unit lattice constants of a = 49.7, b = 69.2, c = 166 Å, α = 89.9, β = 90.0, and γ = 90.0°. Because there was a possibility that the crystal belonged to monoclinic or orthorhombic space group, we reexamined the extinction rule and tried to perform refinement under condition of monoclinic or orthorhombic space group. As a result, the correct space group was found to be P1 and insufficient decrease in R work was observed in refinement under condition of monoclinic or orthorhombic space group. The structure of Smon0123 (N-18/C-5)/CΔ0S (PDB ID, 5GUB) was used as a search model for molecular replacement. After the molecular replacement, rigid body refinement was performed by using N-and C-terminal domains of Smon0123 (N-18/C-5)/CΔ0S. The refined model contained four monomers in an asymmetric unit while the biological unit was monomer. The final model of the complex was refined to R work of 17.3% and R free of 21.6% up to a resolution of 1.95 Å. Ramachandran plot analysis revealed that 97.9% of residues were in the favored regions and 2.12% were in the additional allowed regions. Statistics of diffraction and refinement data are shown in Table S1.
Overall structure and substrate-binding site of CΔ4S6S-bound Smon0123. We have previously determined the four crystal structures of Smon0123 and clarified the specific interactions between the substrate and protein 8 . Thus, the conformational changes and the classification of Smon0123 in bacterial solute-binding proteins in the ABC importer system will be examined hereafter. In general, solute-binding proteins induce a conformational change via the hinge-bending motion, which is one of the two basic mechanisms of protein flexibility. The hinge-bending motion is characterized by a specific torsion angle that changes the rest of the whole proteins into a rigid body, whereas the other mechanism is shear motion, which is characterized by sliding layered structures over one another 19 . Hundreds of structures of solute-binding proteins have been previously determined using various substrates such as sugars, metal ions, amino acids, and peptides 20,21 . Although Smon0123 shows little sequence similarity with other solute-binding proteins, these constructional features are highly conserved 22,23 . Similar to other solute-binding proteins, Smon0123 has two major N and C domains that are connected via a flexible hinge and that captures the substrates via a mechanism called the "Venus's flytrap" 24 . Each major domain is divided into two subdomains (N1-C1-N2-C2 subdomains) ( Fig. 2A).
In the complex crystal structure of Smon0123 with CΔ4S6S, the substrate CΔ4S6S was bound to a cleft between the N and C domains. Far from the cleft, a calcium ion was bound to the C1 subdomain. The electron density of N-terminal 9 residues (Met1Lys19-Gly26) was invisible because of structural flexibility.  Table 1). The cleft had spatial allowance around the two sulfate groups of CΔ4S6S. Two positively charged residues, Arg204 and Arg393, were directly bound to the sulfate group at the C-4 position. Lys210 directly formed hydrogen bonds with the sulfate group at the C-6 position. Therefore, these basic residues were concertedly situated around the negatively charged substrate, causing a positively charged binding site (Fig. 2C). Moreover, aromatic residues such as Tyr146 and Trp284 interacted with the pyranose ring of the substrate. The number of hydrogen bonds with GalNAc4S6S was more than those with ΔGlcUA. In contrast, the number of van der Waals contacts with GalNAc4S6S was less than those with ΔGlcUA.

Conformational changes by substrate binding. To draw a comparison between substrate-free
Smon0123 and CΔ4S6S-bound Smon0123, the torsion angle differences of the Cα backbone between the two forms were plotted (Fig. 3). At both ϕ and ψ plots, structural changes occurred in the hinge region at three loops: Tyr146-Ser154 (N1-C1), Arg319-Thr324 (C1-N2), and Ala414-Ser415 (N2-C2). In particular, the only loop (Arg319-Thr324) that extended over the N and C domains showed a remarkable change, indicating that this loop pulled both the domains. Moreover Smon0123 was approximately 6.9 Å. Trp284 formed a hydrogen bond only with the sulfate group at the C-4 position of CΔ4S6S, although all substrates (CΔ0S, CΔ4S, CΔ6S, and CΔ4S6S) were bound to Trp284 by van der Waals contacts. Ser287 formed hydrogen bonds with hydroxyl groups at the C-6 position of ΔGlcUA in all substrates. All these amino acid residues (Arg204, Lys210, Trp284, and Ser287) belonged to the C1 subdomain, whereas the other amino acid residues located in the substrate-binding site belonged to the N1 or N2 subdomains (Fig. 2B). Therefore, two sulfate groups in CΔ4S6S shifted the arrangements of amino acid residues in the C1 subdomain, resulting in conformational changes in Smon0123/CΔ4S6S owing to the movement of the C domain. Moreover, Smon0123 showed lower affinity with CΔ4S6S than the other chondroitin disaccharides (CΔ0S, CΔ4S, and CΔ6S), suggesting the influence of the interactions among the four subdomains on the affinity level with substrates.  (Fig. 6E). The ATPase activity in the presence of cellobiose as a negative control was comparable with that in the absence of disaccharides (PLS, proteoliposome), while the ATPase activity in the presence of non-sulfated CΔ0S, monosulfated CΔ4S and CΔ6S, and disulfated CΔ4S6S were all enhanced compared to PLS. Although the ATPase activity in the presence of CΔ6S and CΔ4S6S was comparable, the activity in the presence of CΔ4S6S was lowest among these unsaturated chondroitin disaccharides, suggesting that CΔ4S6S bound to Smon0123 in the 39°-closed conformation was possibly unpreferable for ABC transporter in comparison with other GAG disaccharides bound to Smon0123 in the 47°-closed conformation.

Discussion
Solute-binding proteins were previously classified as six clusters on the basis of the features of their three-dimensional structures 20 . A new 7 th cluster has been more recently proposed on the basis of structural features, with relatively a large molecular mass, and EF hand-like calcium-binding sites, which were significantly different from other clusters 21,27 . The members of the 7 th cluster contain only four proteins, namely two alginate-binding AlgQ1 and AlgQ2 in Sphingomonas sp. A1, uncharacterized Blon_2351 in Bifidobacterium logum, and fructooligosaccharide-binding FusA in Streptococcus pneumoniae. The EF hand motif, which is the most common calcium-binding motif in proteins, is usually formed by a helix-loop-helix structure. Calcium ion is usually bound by seven ligands at the vertices of a pentagonal bipyramid 28 . Several atypical motifs that vary in length and secondary structure are called EF hand-like motifs 27 . Because of a large molecular size (57 kDa) and EF hand-like calcium-binding site, Smon0123 should also be added to the 7 th cluster. Seven oxygen atoms of Asp189, Asn191, Asn193, Lys195, Asp197, and Glu198 of Smon0123 were coordinated with the calcium ion. Although the EF hand-like calcium-binding site had no effect on the substrate binding owing to the remote distance, the motif in the alginate-binding AlgQ2 interacts with the ABC transporter for alginate import in the crystal structure 10 , suggesting its role for substrate translocation from the solute-binding protein to the ABC transporter 27 . Solute-binding proteins form open and closed conformations in equilibrium even in the absence of substrates 29 . The cleft is opened up to the solvent to allow substrate to freely bind and dissociate, causing flexibility of the structure 30 . Some studies have reported that the crystal structures of substrate-free binding proteins such as leucine-binding protein 29 , allose-binding protein 30 , galactose-binding protein 31 , and ribose-binding protein 32 form a range of open conformation. However, once the protein recognizes the substrates and closes its domains, the reopening of the protein should be severely restricted because the substrate-bound protein needs to form a complex with the ABC transporter and transfer the substrate. The substrate first binds to a domain of the protein, followed by contact with another domain owing to thermal fluctuations and forms additional contacts to stabilize the closed conformation 19 . Therefore, substrate-bound conformations show much less variations 29 . The glucose-binding protein of Pseudomonas putida forms both glucose and galactose-bound structures, and these two complex structures adopt the closed conformation by the hinge-bending motion, although the magnitude of the hinge-bending motions between the two structures are identical 33 . Furthermore, AlgQ1 and AlgQ2, which  are homologous to Smon0123, exhibit no structural changes depending on various oligoalginates with different constituent sugars and/or polymerization degrees 34,35 . Conversely, the structures of some solute-binding proteins assumed a different hinge-bending motion even in the presence of same substrates, probably because of the The magnitude of the hinge-bending motion in the crystal structure was unexpectedly determined to be 47°; however, C∆4S was bound to Smon0123 instead of CΔ4S6S, which was used as a ligand. This is possibly because of the release of the sulfate group at the C-6 position from CΔ4S6S during crystallization or the presence of contaminated CΔ4S in CΔ4S6S. No sulfatase activity of Smon0123 was also examined by thin-layer chromatography (Fig. S1).
The alginate ABC transporter in Sphingomonas sp. A1 comprises four subunits of the transmembrane domain (AlgM1-AlgM2) and ATP-binding domain (AlgS-AlgS). The complex structure of the alginate ABC transporter (AlgM1-AlgM2/AlgS-AlgS) with the alginate-binding protein AlgQ2 indicates the interaction mode between the binding protein and the ABC transporter 10 . In particular, the helix 5c of AlgM2 is crucial for the interaction with AlgQ2. Each subunit of the alginate ABC transporter shows >45% sequence identities with that of the Streptobacillus one ( To confirm this hypothesis, we investigated the ATPase activity of the ABC transporter in the presence of Smon0123 and CΔ4S6S (Fig. 6E). As a result, the enhancement of its ATPase activity in the presence of CΔ4S6S was lower than that of CΔ0S and CΔ4S, suggesting that CΔ4S6S-bound Smon0123 in the 39°-closed conformation is unpreferable for docking with the ABC transporter compared to that in the 47°-closed conformation. Therefore, Smon0123 is suggested to select preferable substrates by two steps, based on whether to bind and how to bind. Alternative substrate-bound conformation probably prevents the import of the unfavorable substrate CΔ4S6S.
In conclusion, Smon0123 adopts two 39°-and 47°-closed conformations depending on the sulfate group(s) in the substrate by the hinge-bending motion. The two sulfate groups in CΔ4S6S shift the arrangements of the substrate-binding residues in the C1 subdomain, followed by a dynamic conformational change.

Materials and Methods
Materials. CΔ4S6S was purchased from Dextra Laboratories, and chondroitin sulfate A was purchased from Wako Pure Chemical Industries. Columns for metal affinity chromatography (TALON), anion exchange chromatography (Toyopearl DEAE-650M), and gel filtration chromatography (Hi Load 16/60 Superdex 200 pg) were purchased from Clontech, Tosoh Bioscience LLC, and GE Healthcare, respectively. JBScreen Classic 3 for the crystallization kit was purchased from Jena Bioscience. S. moniliformis DSM 12112 was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen. All other analytical grade chemicals used in this study were commercially available.
Plate method for detecting GAG degradation. Bacterial cells were cultured on the following medium plates (ϕ = 90 mm) solidified with 1% agar that contained 1% BSA and 0.2% chondroitin sulfate A, followed by the addition of 1 ml of 2 M acetic acid on the plate. Pedobacter heparinus and Escherichia coli were used as the positive and negative controls for GAG degradation, respectively. S. moniliformis cells were cultured in 0.8% nutrient broth (0.3% beef extract and 0.5% peptone) and 20% horse serum at 37 °C under 5% CO 2 ; P. heparinus cells were cultured in 0.8% nutrient broth at 30 °C, and E. coli cells were cultured in Luria broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) 40  The measurement parameters were as follows: excitation band width, 1 nm; emission band width, 10 nm; response, 2 s; sensitivity, high; excitation wavelength, 280 nm; start to end emission wavelength, 300-500 nm; data pitch, 1 nm; and scan speed, 100 nm/min. The ratio of change in fluorescence intensity in the presence of CΔ4S6S compared with that in the absence of CΔ4S6S was plotted, and the dissociation constant (K d value) was determined.
X-ray crystallography. The purified Smon0123 (N-18/C-5) was crystallized by sitting drop vapor diffusion. In 96-well sitting drop plates, 1 μl of 10.8 mg/ml Smon0123 (N-18/C-5) and an equal volume of reservoir solution for crystallization were mixed in the presence of 0.5 mM CΔ4S6S. The solution was kept at 20 °C for 2 weeks until the crystals sufficiently grew. A single crystal was picked up using a nylon loop from the drop, soaked in a reservoir solution that contained 20% ethylene glycol, and instantly frozen using cold nitrogen gas. Synchrotron radiation X-ray irradiated the crystal at 1.00 Å wavelength, and X-ray diffraction data were collected using the MAR225HE (Rayonix) detector at BL-26B1 beamline in SPring-8 (Harima, Japan). The obtained data were indexed, integrated, and scaled using the HKL-2000 program 41 . Some crystals in the reservoir solution that contained 18% (w/v) PEG 4000, 20% (w/v) isopropanol, and 0.1 M Na HEPES (pH 7.5) from the JBScreen Classic 3 crystallization kit gave high-resolution diffraction data. The structure was determined through molecular replacement by Molrep in the CCP4 program 42  Similarly, its crystal structure was determined through molecular replacement, although the protein was found to interact with C∆4S but not C∆4S6S in the cleft.

Magnitude of hinge-bending motion.
To calculate the magnitude of the hinge-bending motion, N domains of substrate-free and substrate-bound Smon0123 were superimposed. By using FIT program, rotation matrix, translation vectors, rotation angles, and screwing distances of C domains of substrate-free and substrate-bound Smon0123 were calculated from centers of gravity.
ATPase assay. As previously described 8