Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA; The UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, San Francisco, CA 94158, USA.
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA 01605, USA; School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India.
Cell Syst. 2023 Mar 15;14(3):237-246.e7. doi: 10.1016/j.cels.2023.01.003. Epub 2023 Feb 17.
Allosteric regulation is central to protein function in cellular networks. A fundamental open question is whether cellular regulation of allosteric proteins occurs only at a few defined positions or at many sites distributed throughout the structure. Here, we probe the regulation of GTPases-protein switches that control signaling through regulated conformational cycling-at residue-level resolution by deep mutagenesis in the native biological network. For the GTPase Gsp1/Ran, we find that 28% of the 4,315 assayed mutations show pronounced gain-of-function responses. Twenty of the sixty positions enriched for gain-of-function mutations are outside the canonical GTPase active site switch regions. Kinetic analysis shows that these distal sites are allosterically coupled to the active site. We conclude that the GTPase switch mechanism is broadly sensitive to cellular allosteric regulation. Our systematic discovery of new regulatory sites provides a functional map to interrogate and target GTPases controlling many essential biological processes.
变构调节是细胞网络中蛋白质功能的核心。一个基本的开放性问题是，细胞对变构蛋白的调节是仅发生在少数几个定义明确的位置，还是发生在整个结构中分布的许多位点。在这里，我们通过在天然生物网络中进行深度诱变，以残基分辨率探测 GTPase-蛋白开关（控制通过调节构象循环进行的信号转导的蛋白质）的调节。对于 GTPase Gsp1/Ran，我们发现 4315 个测定突变中有 28%表现出明显的功能获得响应。在功能获得突变富集的六十个位置中有二十个位于经典 GTPase 活性位点开关区域之外。动力学分析表明，这些远端位点与活性位点呈变构耦联。我们得出结论，GTPase 开关机制对细胞变构调节具有广泛的敏感性。我们系统地发现新的调节位点为研究和靶向控制许多重要生物过程的 GTPase 提供了功能图谱。
