Site-Specific Characterization of the Association of Xylooligosaccharides with the CBM13 Lectin-like Xylan Binding Domain from Streptomyces lividans Xylanase 10A by NMR Spectroscopy

Abstract

Endo-beta-1,4-xylanase 10A (Xyn10A) from Streptomyces lividans includes an N-terminal catalytic module and a 130-residue C-terminal family 13 carbohydrate-binding module (CBM13). This latter domain adopts a beta-trefoil structure with three potential binding sites (alpha, beta, and gamma) for a variety of small sugars, xylooligosaccharides, and xylan polymers. To investigate the role of this multivalency in carbohydrate binding, we have used NMR spectroscopy to characterize the interaction of isolated CBM13 with a series of sugars. We have assigned resonances from the main chain nuclei of CBM13 using heteronuclear NMR experiments. Analysis of (15)N NMR relaxation data using the extended model free formalism reveals that CBM13 tumbles as an oblate ellipsoid (D( parallel)/D( perpendicular) = 0.80 +/- 0.02) and that its backbone is relatively rigid on the sub-nanosecond time scale. In particular, the three binding sites show no distinct patterns of increased internal mobility. Ligand-induced chemical shift changes in the (1)H-(15)N HSQC spectra of CBM13 were monitored as a function of increasing concentrations of L-arabinose, lactose, D-xylose, xylobiose, xylotetraose, and xylohexaose. Patterns of shift perturbations for well-resolved resonances demonstrate that all of these sugars associate independently with the three binding sites of CBM13. On the basis of the site-specific association constants derived from a quantitative analysis of these titration data, we show that L-arabinose, lactose, and D-xylose preferentially bind to the alpha site of CBM13, xylobiose binds equally well to all three sites, and xylotetraose and xylohexaose prefer binding to the beta site. Inspection of the crystallographic structure of CBM13 [Notenboom, V., Boraston, A. B., Williams, S. J., Kilburn, D. G., and Rose, D. R. (2002) Biochemistry 41, 4246-4254] provides a rationalization for these results.

Crystal Structures of Decorated Xylooligosaccharides Bound to a Family 10 Xylanase from Streptomyces olivaceoviridis E-86

“…In the SoXyn10A⅐X2 and SoXyn10A⅐X3 complexes, continuous electron densities were observed on both sides of the bound xylose at the O-1 and O-4 atoms, and the SoCBM13 appeared to bind to internal sites of xylan. The crystal structure and NMR spectroscopy of CBM13 from S. lividans also revealed similar structural features (20,21).…”

The Family 6 Carbohydrate Binding Module CmCBM6-2 Contains Two Ligand-binding Sites with Distinct Specificities

“…CBM13s are members of the "ricin-superfamily" of lectins that have a ␤-trefoil fold (36 -38). These proteins have three distinct sugar-binding sites that are all able to bind to the same monoand oligosaccharide ligands, although the actual affinities for these molecules can vary between the different sites (39). CBM20s contain two discrete starch-binding sites that display very similar ligand specificities (40).…”

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…The fold of the resulting molecule has a pseudo-3-fold axis [45,46]. The 3-fold symmetry is amenable to the presence of functional carbohydrate-binding sites in each of the three trefoil subdomains, which is exploited by the CBM13 modules of Streptomyces lividans and Streptomyces olivaceoviridis xylanases in order to maintain a reasonably high affinity for β-1,4-linked polymers of xylose [47][48][49][50]. The plant lectins with this fold, e.g.…”

“…This was first proposed for the family 13 CBM from S. lividans xylanase 10A on the basis of its similarity to the multivalent ricin toxin B-chain. It was subsequently demonstrated by mutagenesis [47], NMR [49] and X-ray crystallography [48] that this module had three separate binding sites, one in each of its trefoil subdomains ( Figure 8A). The presence of multiple binding sites enabled the module to interact simultaneously with multiple binding sites within polymerized xylose to enhance its overall affinity by approx.…”