Abstract:
The receptor for thyroid stimulating hormone (TSHR), a GPCR, is the primary antigen in autoimmune hyperthyroidism (Graves\' disease) caused by stimulating TSHR antibodies. While we have previously published a full length model of the TSHR, including its leucine rich domain (LRD), linker region (LR) and transmembrane domain (TMD), to date, only a partial LRD (aa 21-261) stabilized with TSHR autoantibodies has been crystallized. Recently, however, cryo-EM structures of the full-length TSHR have been published but they include only an incomplete LR. We have now utilized the cryo-EM models, added disulfide bonds to the LR and performed longer (3000 ns) molecular dynamic (MD) simulations to update our previous model of the entire full-length TSHR, with and without the presence of TSH ligand. As in our earlier work, the new model was embedded in a lipid membrane and was solvated with water and counterions. We found that the 3000 ns Molecular Dynamic simulations showed that the structure of the LRD and TMD were remarkably constant while the LR, known more commonly as the \"hinge region\", again showed significant flexibility, forming several transient secondary structural elements. Analysis of the new simulations permitted a detailed examination of the effect of TSH binding on the structure of the TSHR. We found a structure-stabilizing effect of TSH, including increased stability of the LR, which was clearly demonstrated by analyzing several intrinsic receptor properties including hydrogen bonding, fluctuation of the LRD orientation, and radius of gyration. In conclusion, we were able to quantify the flexibility of the TSHR and show its increased stability after TSH binding. These data indicated the important role of ligands in directing the signaling structure of a receptor.
摘要:
促甲状腺激素受体(TSHR),aGPCR,是由刺激TSHR抗体引起的自身免疫性甲状腺功能亢进(Graves病)的主要抗原。虽然我们之前已经发布了TSHR的全长模型,包括其富含亮氨酸的结构域(LRD),连接区(LR)和跨膜结构域(TMD),到目前为止,只有用TSHR自身抗体稳定的部分LRD(aa21-261)已经结晶。最近,然而,全长TSHR的cryo-EM结构已经发表,但它们仅包括不完整的LR。我们现在已经使用了低温EM模型,将二硫键添加到LR,并进行更长的(3000ns)分子动力学(MD)模拟,以更新我们以前的整个全长TSHR模型,在存在和不存在TSH配体的情况下。和我们之前的工作一样,新模型嵌入脂质膜中,用水和反离子溶剂化。我们发现,3000ns分子动力学模拟表明,LRD和TMD的结构非常恒定,而LR,通常称为“铰链区”,再次显示出显著的灵活性,形成几个瞬态次级结构元素。新模拟的分析允许详细检查TSH结合对TSHR结构的影响。我们发现TSH的结构稳定作用,包括增加LR的稳定性,通过分析包括氢键在内的几种内在受体特性清楚地证明了这一点,LRD方向的波动,和回转半径。总之,我们能够量化TSHR的灵活性,并显示其在TSH结合后的稳定性增加。这些数据表明配体在指导受体的信号传导结构中的重要作用。