Acta Crystallographica Section D

Biological Crystallography

Volume 61, Part 1 (January 2005)

research papers

Acta Cryst. (2005). D61, 22-32 [ doi:10.1107/S0907444904025715 ]

Binding of N-acetylglucosamine oligosaccharides to hen egg-white lysozyme: a powder diffraction study

R. B. Von Dreele

Abstract: The binding of N-acetylglucosamine oligosaccharides (NAG_n, n = 2-6) to hen egg-white lysozyme (HEWL; EC 3.2.1.17) was investigated by X-ray powder diffraction at room temperature. Each NAG_n examined was found to bind to lysozyme in rapid-precipitation preparations in 1.0 M NaCl pH 6.0 buffer. The location of each NAG_n was easily found from difference Fourier maps generated from structure factors extracted during preliminary Rietveld refinements. Full NAG_n-protein structures were subjected to combined Rietveld and stereochemical restraint refinements (R_wp = 2.28-2.59%; R_p = 1.81-2.04%; $R_{\rm F}^{2}$ = 3.91-5.80%) and revealed binding modes for NAG_n that depended on the length of the NAG oligosaccharide. The NAG₂ ligand was found in the BC sites in the cleft of HEWL, NAG₃ was found to bind in both the ABC and BCD sites in the ratio 35:65 and NAG₄ and NAG₅ bound to the ABCD and ABCDE sites, respectively, while NAG₆ only bound to sites ABCDE, leaving the F site empty with the remaining saccharide ring located in a solvent region adjacent to the A site. All protein powder diffraction patterns in this study consisted of extremely sharp Bragg peaks consistent with 1 µm crystallites that were devoid of line-broadening defects. Details of the stereochemical restraints used in these refinements and their impact on structural validation are also discussed.

PDB references: 1sf4, 1sf6, 1sf7, 1sfb and 1sfg

Keywords: powder diffraction; N-acetylglucosamine oligosaccharides; lysozyme; structure validation.