Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva, 8410501, Israel.
Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Sci Rep. 2022 May 23;12(1):8657. doi: 10.1038/s41598-022-12731-2.
The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we present a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The yeast detergent-resistant cell wall presents a unique opportunity for compartmentalization, to physically link the receptor's phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method reveals a surprising amenability of the GPCR scaffold to stabilization, and suggests an intriguing possibility of amending the stability properties of GPCR by varying the structural status of the C-terminus.
由于阐明这些极不稳定的膜蛋白的结构存在巨大困难，G 蛋白偶联受体（GPCR）作为药物发现靶点的巨大潜力尚未得到充分实现。现有的 GPCR 稳定性工程方法既繁琐又低通量；此外，能够从这些技术中受益的 GPCR 范围有限。在这里，我们提出了一个基于酵母的筛选平台，用于一步分离在短链去污剂存在下稳定的 GPCR 变体，这是使用气相扩散法成功结晶所必需的特征。酵母去污剂抗性细胞壁为分隔提供了独特的机会，将受体的表型与其编码 DNA 物理连接起来，从而以前所未有的效率发现稳定的 GPCR 变体。该方法鉴定的突变范围表明 GPCR 支架具有惊人的稳定性，并且通过改变 C 末端的结构状态来改变 GPCR 的稳定性特性的可能性令人着迷。
