Garza-Garcia Acely, Esposito Diego, Rieping Wolfgang, Harris Richard, Briggs Cherry, Brown Marion H, Driscoll Paul C
Division of Molecular Structure, National Institute for Medical Research, Medical Research Council, The Ridgeway, Mill Hill, London NW7 1AA, UK.
J Mol Biol. 2008 Apr 18;378(1):129-44. doi: 10.1016/j.jmb.2008.02.006. Epub 2008 Feb 12.
The lymphocyte receptor CD5 influences cell activation by modifying the strength of the intracellular response initiated by antigen engagement. Regulation through CD5 involves the interaction of one or more of its three scavenger receptor cysteine-rich domains present in the extracellular region. Here, we present the 3D solution structure of a non-glycosylated double mutant of the N-terminal domain of human CD5 expressed in Escherichia coli (eCD5d1m), which has enhanced solubility compared to the non-glycosylated wild-type (eCD5d1). In common with a glycosylated form expressed in Pichia pastoris, the [(15)N,(1)H]-correlation spectra of both eCD5d1 and eCD5d1m exhibit non-uniform temperature-dependent signal intensities, indicating extensive conformational fluctuations on the micro-millisecond timescale. Although approximately one half of the signals expected for the domain are absent at 298 K, essentially complete resonance assignments and a solution structure could be obtained at 318 K. Because of the sparse nature of the experimental restraint data and the potentially important contribution of conformational exchange to the nuclear Overhauser effect peak intensity, we applied inferential structure determination to calculate the eCD5d1m structure. The inferential structure determination ensemble has similar features to that obtained by traditional simulated annealing methods, but displays superior definition and structural quality. The eCD5d1m structure is similar to other members of the scavenger receptor cysteine-rich superfamily, but the position of the lone alpha helix differs due to interactions with the unique N-terminal region of the domain. The availability of an experimentally tractable form of CD5d1, together with its 3D structure, provides new tools for further investigation of its function within intact CD5.
淋巴细胞受体CD5通过改变抗原结合引发的细胞内反应强度来影响细胞活化。通过CD5的调节涉及细胞外区域中存在的其三个富含半胱氨酸的清道夫受体结构域中的一个或多个的相互作用。在这里，我们展示了在大肠杆菌中表达的人CD5 N端结构域的非糖基化双突变体（eCD5d1m）的三维溶液结构，与非糖基化野生型（eCD5d1）相比，其溶解性增强。与在毕赤酵母中表达的糖基化形式一样，eCD5d1和eCD5d1m的[(15)N,(1)H]相关光谱均表现出非均匀的温度依赖性信号强度，表明在微毫秒时间尺度上存在广泛的构象波动。尽管在298 K时该结构域预期的信号约有一半缺失，但在318 K时基本上可以获得完整的共振归属和溶液结构。由于实验约束数据的稀疏性以及构象交换对核Overhauser效应峰强度的潜在重要贡献，我们应用推理结构测定来计算eCD5d1m结构。推理结构测定集合具有与传统模拟退火方法获得的集合相似的特征，但显示出更好的清晰度和结构质量。eCD5d1m结构与富含半胱氨酸的清道夫受体超家族的其他成员相似，但由于与该结构域独特的N端区域相互作用，单个α螺旋的位置有所不同。可实验处理的CD5d1形式及其三维结构的可用性为进一步研究其在完整CD5中的功能提供了新工具。
