Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA; Present Address: Department of Basic Pharmaceutical Sciences, Fred Wilson School of Pharmacy, High Point University, High Point, NC, 27268, USA.
School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.
J Mol Biol. 2020 Mar 27;432(7):1996-2014. doi: 10.1016/j.jmb.2020.01.028. Epub 2020 Feb 6.
The class B G protein-coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N-glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin-bound, GlcNAc-bearing CTR ECD at 1.78 and 2.85 Å resolutions and analyze the mechanism of the glycan effect. The N130 GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone-bound, N-glycan-free ECD, which suggested that the GlcNAc might affect CTR dynamics not observed in the static crystallographic snapshots. Hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations revealed that glycosylation stabilized a β-sheet adjacent to the N130 GlcNAc and the N-terminal α-helix near the peptide-binding site while increasing flexibility of the peptide-binding site turret loop. These changes due to N-glycosylation increased the ligand on-rate and decreased its off-rate. The glycan effect extended to RAMP-CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N-glycosylation can modulate GPCR function by altering receptor dynamics.
B 类 G 蛋白偶联受体 (GPCR) 降钙素受体 (CTR) 是骨质疏松症和糖尿病的药物靶点。其细胞外结构域 (ECD) 中天门冬酰胺 130 的 N-糖基化增强了降钙素激素与近端 GlcNAc 残基的亲和力，这种效应通过未知机制介导。在这里，我们呈现了两个三文鱼降钙素结合、带有 GlcNAc 的 CTR ECD 的晶体结构，分辨率分别为 1.78 和 2.85Å，并分析了糖基化效应的机制。N130 的 GlcNAc 不与激素接触。令人惊讶的是，这些结构与没有 N-聚糖的激素结合的 ECD 结构几乎相同，这表明 GlcNAc 可能会影响 CTR 动力学，而这些动力学在静态晶体学快照中观察不到。氢氘交换质谱和分子动力学模拟表明，糖基化稳定了与 N130 GlcNAc 相邻的β-折叠以及靠近肽结合位点的 N 端α-螺旋，同时增加了肽结合位点塔环的灵活性。这些由于 N-糖基化引起的变化增加了配体的结合速率，降低了其解离速率。糖基化效应扩展到 RAMP-CTR 淀粉样肽受体复合物，并且在相关的 CGRP 受体中也得到保守。这些结果表明，N-糖基化可以通过改变受体动力学来调节 GPCR 功能。
