title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3. mcintosh@otter.biochem.ubc.ca
authors.forename: Manuela
authors.initials: M
authors.lastname: Schärpf
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Connelly
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Lee
authors.forename: Alisdair B
authors.initials: AB
authors.lastname: Boraston
authors.forename: R Antony J
authors.initials: RA
authors.lastname: Warren
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D001089:Arabinose / Q000737:chemistry; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007785:Lactose / Q000737:chemistry; D037102:Lectins / Q000737:chemistry*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000737:chemistry*; D013997:Time Factors; D043325:Xylan Endo-1,3-beta-Xylosidase; D014994:Xylose / Q000737:chemistry; D014995:Xylosidases / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D014994:Xylose; D001089:Arabinose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D007785:Lactose