Muraki M, Harata K, Sugita N, Sato K i
Biomolecules Department, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305, Japan.
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):834-43. doi: 10.1107/s090744499800122x.
Human lysozyme (HL) labelled with the 2',3'-epoxypropyl beta-glycoside of Man-beta1,4-GlcNAc was crystallized at pH 4.5. The cell dimensions were a = 36.39, b = 116.38, c = 30.91 A and the space group was P212121. The unit cell contained four molecules (Vm = 2.18 A3 Da-1). The crystal structure was determined by molecular replacement and refined to an R value of 0.168 for 7060 reflections [|Fo| > 3sigma(F)] in the resolution range 8.0-2.1 A. A prominent shift of the Calpha-atom positions by up to 3.8 A in the region of residues 45-50 was observed compared with wild-type HL. Owing to the conformational change in this region the intermolecular contacts were altered remarkably compared to wild-type HL, explaining the difference in molecular packing. The Man-beta1,4-GlcNAc moiety occupied subsites B and C in the substrate-binding site of HL. Several differences in the hydrogen-bonded contacts between the ligand part and the protein part were observed for HL labelled with the 2',3'-epoxypropyl beta-glycoside of Man-beta1,4-GlcNAc compared with HL labelled with the corresponding derivatives of GlcNAc-beta1, 4-GlcNAc and Gal-beta1,4-GlcNAc. In contrast to the replacement of GlcNAc with Gal, the replacement of GlcNAc with Man did not sacrifice the stacking interactions with the side-chain group of Tyr63 as determined by the parallelism of the apolar face of the carbohydrate residue and the aromatic plane of the Tyr63 side chain. The 2',3'-epoxypropyl beta-glycoside of Man-beta1,4-GlcNAc exhibited almost the same affinity towards HL as Gal-beta1,4-GlcNAc, a much lower affinity than that of GlcNAc-beta1,4-GlcNAc. The difference in the protein-ligand interactions was discussed in relation to the carbo-hydrate-residue recognition specificity at subsite B of HL. The results suggested that Gln104 was a determinant for the strong recognition of GlcNAc residue at subsite B in HL.
用Man-β1,4-GlcNAc的2',3'-环氧丙基β-糖苷标记的人溶菌酶（HL）在pH 4.5条件下结晶。晶胞参数为a = 36.39、b = 116.38、c = 30.91 Å，空间群为P212121。晶胞包含四个分子（Vm = 2.18 Å3 Da-1）。通过分子置换法确定了晶体结构，并对分辨率范围为8.0 - 2.1 Å的7060个反射（|Fo| > 3σ(F)）进行精修，R值为0.168。与野生型HL相比，在45 - 50位残基区域观察到Cα原子位置有高达3.8 Å的显著位移。由于该区域的构象变化，与野生型HL相比，分子间接触发生了显著改变，这解释了分子堆积的差异。Man-β1,4-GlcNAc部分占据了HL底物结合位点的B和C亚位点。与用GlcNAc-β1,4-GlcNAc和Gal-β1,4-GlcNAc的相应衍生物标记的HL相比，观察到用Man-β1,4-GlcNAc的2',3'-环氧丙基β-糖苷标记的HL在配体部分和蛋白质部分之间的氢键接触存在若干差异。与用Gal取代GlcNAc不同，用Man取代GlcNAc并没有像通过碳水化合物残基的非极性面与Tyr63侧链的芳香平面的平行度所确定的那样牺牲与Tyr63侧链基团的堆积相互作用。Man-β1,4-GlcNAc的2',3'-环氧丙基β-糖苷对HL的亲和力与Gal-β1,4-GlcNAc几乎相同，远低于GlcNAc-β1,4-GlcNAc。结合HL的B亚位点的碳水化合物残基识别特异性讨论了蛋白质 - 配体相互作用的差异。结果表明，Gln104是HL中B亚位点对GlcNAc残基进行强识别的决定因素。
