Jouravel Natalia, Sablin Elena, Togashi Marie, Baxter John D, Webb Paul, Fletterick Robert J
Department of Biochemistry and Biophysics, University of California San Francisco (UCSF), San Francisco, California 94158, USA.
Proteins. 2009 Apr;75(1):111-7. doi: 10.1002/prot.22225.
Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.
在用于X射线晶体学的蛋白质纯化过程中，蛋白质的质量和稳定性至关重要。一种易于操作和结晶的目标蛋白质成为了一种有价值的产物，可用于药物设计与发现的高通量晶体学研究。在这项工作中，一个单一的表面突变D355R被证明对于将人甲状腺激素受体（TR LBD）适度稳定的单体配体结合结构域转化为稳定的二聚体至关重要。利用X射线晶体学解析了D335R TR LBD突变体的结构，并将其精修至2.2 Å分辨率，R（free）/R值为24.5/21.7。晶体不对称单元揭示了TR二聚体，其中两个激素结合的LBD分子通过二重对称相关。两个LBD之间的离子界面包括H10-H11环和H6-H7环内的残基以及两个原体的螺旋8的C端一半。一个TR分子引入的残基Arg 355与第二个分子的Glu 324之间形成的直接分子间接触成为了1330 Ų扩展二聚化界面的一部分，该界面是强复合物组装的特征，缓冲溶质进一步增强了这种组装。
