Bundle D R, Baumann H, Brisson J R, Gagné S M, Zdanov A, Cygler M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Biochemistry. 1994 May 3;33(17):5183-92. doi: 10.1021/bi00183a023.
NMR and crystallography have been used to study antigen conformational changes that occur in a trisaccharide-Fab complex in solution and in the solid state. NOE buildup rates from transferred NOE experiments show that the antigenic determinant of a Salmonella lipopolysaccharide, represented by the trisaccharide methyl glycoside alpha-D-Galp(1-->2 [alpha-D-Abep(1-->3)]- alpha-D-Manp1-->OMe (1), undergoes a protein-induced conformational shift about the Gal-->Man glycosidic linkage when it is bound by a monoclonal antibody in aqueous solution. The same trisaccharide was crystallized with Fab, and a solved structure at 2.1-A resolution revealed that the conformation of the trisaccharide ligand was similar to that seen in a dodesaccharide-Fab complex [Cygler et al. (1991) Science 253, 442-445), where the Gal-Man linkage also experienced a similar conformational shift. Distance constraints derived from the TRNOE buildup curves are consistent with two bound trisaccharide conformations, one of which correlates with the ligand conformation of the crystalline Fab-trisaccharide complex. In this bound conformation, short interatomic distances between Abe O-2 and Gal O-2 permit an oligosaccharide intramolecular hydrogen bond. Despite its relatively low energy, a preponderance of this conformer could not be detected in aqueous or DMSO solutions of free trisaccharide by either 1H or 13C NMR experiments. In DMSO, a different intramolecular hydrogen bond between Abe O-2 and Man O-4 was observed due to a solvent-induced shift in the conformational equilibria (relative to aqueous solution). Molecular modeling of the trisaccharide in the binding site and as the free ligand suggested that the protein imposes an induced fit on the antigen, primarily resulting in a shift of the Gal-Man phi torsional angle. This reduces the interproton separation between Abe H-3 and Gal H-1 with a marked increase in the intensity of the previously weak NOEs between the protons of the noncovalently linked galactose and abequose residues. The impact of the conformational shift on gross trisaccharide topology is sufficiently small that binding modes inferred from functional group replacements are not impaired.
核磁共振（NMR）和晶体学已被用于研究在溶液和固态中三糖 - Fab复合物中发生的抗原构象变化。转移NOE实验的NOE积累速率表明，由三糖甲基糖苷α - D - Galp(1→2 [α - D - Abep(1→3)] - α - D - Manp1→OMe（1）代表的沙门氏菌脂多糖的抗原决定簇，在水溶液中与单克隆抗体结合时，围绕Gal→Man糖苷键发生蛋白质诱导的构象转变。相同的三糖与Fab一起结晶，并且在2.1 Å分辨率下解析的结构表明，三糖配体的构象与在十二糖 - Fab复合物中看到的构象相似[Cygler等人（1991年）《科学》253，442 - 445]，其中Gal - Man键也经历了类似的构象转变。从TRNOE积累曲线得出的距离限制与两种结合的三糖构象一致，其中一种与结晶的Fab - 三糖复合物的配体构象相关。在这种结合构象中，Abe O - 2和Gal O - 2之间的短原子间距离允许寡糖分子内氢键形成。尽管其能量相对较低，但通过1H或13C NMR实验在游离三糖的水溶液或DMSO溶液中均未检测到这种构象异构体的优势。在DMSO中，由于构象平衡的溶剂诱导转变（相对于水溶液），观察到Abe O - 2和Man O - 4之间存在不同的分子内氢键。结合位点中三糖以及游离配体的分子建模表明，蛋白质对抗原施加诱导契合，主要导致Gal - Man φ扭转角的转变。这减少了Abe H - 3和Gal H - 1之间的质子间距离，同时非共价连接的半乳糖和阿比可糖残基质子之间先前较弱的NOE强度显著增加。构象转变对三糖总体拓扑结构的影响足够小，以至于从官能团取代推断的结合模式不受损害。
