Structural insights into asymmetric activation of the calcium-sensing receptor-Gq complex.
Structural insights into asymmetric activation of the calcium-sensing receptor-Gq complex.
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DOI：
10.1038/s41422-023-00892-2
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发表时间：
2023-11
期刊：
影响因子：
44.1
通讯作者：
S. Ling;Xianyu Meng;Yuan Zhang;Zhemin Xia;Yingxin Zhou;Fan Yang;P. Shi;Chaowei Shi;Changlin Tian
中科院分区：
文献类型：
--
作者：
S. Ling;Xianyu Meng;Yuan Zhang;Zhemin Xia;Yingxin Zhou;Fan Yang;P. Shi;Chaowei Shi;Changlin Tian
人类钙感应受体（CaSR）是一类CG蛋白偶联受体（GPCR），负责维持血液中Ca2+的稳态。1,2在正常生理过程中，为了响应细胞外刺激，CaSR可以激活包括Gq、Gi或G12/13在内的多种细胞内信号通路。1 CaSR功能障碍可导致高钙血症和低钙血症。3,4作为钙化剂的阳性变构调节剂（Positive allosteric modulators, PAMs），即cinacalcet、evocalcet和etelcalcetide，被开发为治疗慢性肾病患者甲状旁腺功能亢进的药物。最近，已经确定了几种处于不同激活状态的CaSR结构，并提出了不同的激活机制（不对称或对称）。8-12 cinacalet和evocalcet结合的CaSR结构显示了不对称跨膜结构域（TMD），该结构域通过PAM分子以不同姿态结合来稳定。11相反，基于r -568结合和无pam的CaSR活性结构提出了对称活化，它们显示对称的TMD二聚体。9然而，由于缺乏G蛋白偶联复合物结构，CaSR的完整激活和调控机制尚不清楚。近年来，在解码C类gpcr的激活机制方面取得了突破。13-15然而，C类GPCR中的新型gi结合袋是否在不同亚型的G蛋白中保守仍不清楚。在这里，我们报道了CaSR-Gq复合物的低温电镜结构，这是与Gq蛋白偶联的第一类C类GPCR结构。该复合物是在激动剂和cinacalcet存在的情况下，通过NanoBit tethering策略、Gq工程和抗体scFv16的方法进行组装的。CaSR c端LgBiT融合（20-892）对受体活性影响不大（补充信息，图S1）。CaSR-Gq配合物的cro - em结构以3.36的总分辨率求解Å。对单个CaSR和Gq蛋白进行进一步的局部修饰，将分辨率分别提高到3.04 Å和3.07 Å（图1a；补充信息，图S2）。在cinacalcet存在的情况下，测定了单个pam结合CaSR（20-892）的结构，总分辨率为2.7 Å（图1b；补充信息，图S3）。值得注意的是，使用从CaSR- gq复合物样品中选择的颗粒，还获得了pam结合的CaSR (20-892)-LgBit的重建图谱（补充信息，图S2）。pam结合的CaSR (20-892)-LgBit的结构与pam结合的CaSR（20-891）的结构几乎相同，均方根偏差（RMSD）为0.34 Å，表明融合的LgBit对CaSR的构象没有影响。CaSR- gq复合物和pam结合的CaSR的高分辨率图谱使我们能够构建具有大多数残基侧链位置明确的模型，以及配体和pam（补充信息，图2）。S4， S5和表S1)。在CaSR-Gq复合物中，在TM6的细胞内半部分之间也观察到胆固醇和脂质（补充信息，图S6a），这被认为有助于增加二聚体TMD排列的稳定性。gq偶联CaSR的整体结构采用紧凑的封闭构象，类似于激动剂结合或激动剂/ pam结合的CaSR 9-11（图2）。1;补充信息，图S6a)。值得注意的是，在gq偶联CaSR的每个原聚体中，cinacalcet具有不同的位姿；一种采用弯曲构象，另一种采用延伸构象。Gq蛋白对…
Dear Editor, The human calcium-sensing receptor (CaSR) is a class CG proteincoupled receptor (GPCR) responsible for maintaining Ca2+ homeostasis in blood. 1, 2 In normal physiological processes, in response to extracellular stimuli, CaSR can activate multiple intracellular signaling pathways involving Gq, Gi or G12/13. 1 Dysfunctions of CaSR can lead to hypercalcaemic and hypocalcaemic disorders. 3, 4 Positive allosteric modulators (PAMs) serving as calcimimetics, namely, cinacalcet, evocalcet and etelcalcetide, were developed as drugs to treat hyperparathyroidism in patients with chronic kidney disease. 5-7 Recently, several structures of CaSR in distinct activation states have been determined, and different activation mechanisms (asymmetric or symmetric) have been proposed. 8-12 Structures of cinacalcet-and evocalcet-bound CaSR revealed the asymmetric transmembrane domain (TMD), which is stabilized by PAM molecules binding in different poses. 11 In contrast, symmetric activation was proposed based on R-568-bound and PAM-free active structures of CaSR, which display symmetric TMD dimers. 9 However, due to the absence of G protein-coupled complex structure, the complete activation and modulation mechanism of CaSR are still elusive. Recently, breakthroughs have been made in decoding the activation mechanism of class C GPCRs. 13-15 However, whether the novel Gi-binding pocket in class C GPCR is conserved across different subtypes of G proteins also remains unclear. Here, we report the cryo-EM structure of the CaSR-Gq complex, the first class C GPCR structure coupled with Gq protein. The complex was assembled in the presence of agonists and cinacalcet, assisted by approaches of NanoBit tethering strategy, Gq engineering and antibody scFv16. LgBiT fusion at the C-terminus of CaSR (20-892) has little effect on the activity of the receptor (Supplementary information, Fig. S1). The cryo-EM structure of CaSR-Gq complex was solved at an overall resolution of 3.36 Å. Further local refinements of the individual CaSR and Gq protein improved the resolution to 3.04 Å and 3.07 Å, respectively (Fig. 1 a; Supplementary information, Fig. S2). The structure of individual PAM-bound CaSR (20-892) was determined in the presence of cinacalcet, with an overall resolution of 2.7 Å (Fig. 1 b; Supplementary information, Fig. S3). It is noteworthy that a reconstruction map of PAM-bound CaSR (20-892)-LgBit was also obtained using the particles selected from CaSR-Gq complex sample (Supplementary information, Fig. S2). The structure of PAM-bound CaSR (20-892)-LgBit is almost identical to that of PAM-bound CaSR (20-891), with a root mean square deviation (RMSD) of 0.34 Å, demonstrating that the fused LgBit has no effect on the conformation of CaSR. The high-resolution maps of CaSR-Gq complex and PAM-bound CaSR enabled us to build the models with unambiguous placement of side chains for most residues, as well as ligands and PAMs (Supplementary information, Figs. S4, S5 and Table S1). Cholesterol and lipid are also observed between the intracellular halves of TM6 in CaSR-Gq complex (Supplementary information, Fig. S6a), which were suggested to contribute additional stability to the dimeric TMD arrangement. The overall architecture of Gq-coupled CaSR adopts a compact closed conformation, similar to that of agonist-bound or agonist/PAM-bound CaSR 9-11(Fig. 1 a; Supplementary information, Fig. S6a). Notably, the cinacalcet adopted distinct poses in each protomer of Gq-coupled CaSR; one adopted a bent conformation, and the other presented an extended conformation. The Gq protein couples …