Department of Biology, College of Science, United Arab Emirates University, Al Ain PO Box 15551, UAE.
Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands, WA 6009, Australia.
Int J Mol Sci. 2020 Dec 28;22(1):209. doi: 10.3390/ijms22010209.
Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII's k by 2.6 fold with no effect on its B. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.
血红蛋白肽以其在血压控制中的作用而闻名。最近，我们在人胚肾（HEK293）细胞中揭示了 LVV-血红蛋白肽-7（LVV-H7）对血管紧张素 II（AngII）1 型受体（AT1R）的正向调节作用。在这里，我们使用基于纳米荧光素酶的生物发光共振能量转移（NanoBRET）测定法在 HEK293FT 细胞中检查了 LVV-H7 在 AT1R 上的分子结合行为及其对 AngII 结合的影响，以及分子对接和分子动力学（MD）研究。饱和和实时动力学支持 LVV-H7 对 AngII 结合的正向影响。虽然竞争性拮抗剂奥美沙坦与 AngII 结合竞争，但 LVV-H7 略微但显著地将 AngII 的 k 值降低了 2.6 倍，而对其 B 没有影响。分子对接和 MD 模拟表明，LVV-H7 在 AT1R 的细胞内区域的结合以变构方式增强了 AngII 的结合。LVV-H7 靶向 AT1R 的细胞内环 2 和 3 上的残基，这些残基是其他 GPCR 中变构调节剂的已知结合位点。我们的数据证明了 LVV-H7 对 AngII 结合的变构作用，这与先前报道的 AT1R 活性和信号的正向调节一致。这进一步支持了血红蛋白肽对 AT1R 的药理学靶向作用，这对血管和肾脏生理学具有重要意义。
