Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033.
Proc Natl Acad Sci U S A. 2023 Apr 4;120(14):e2219254120. doi: 10.1073/pnas.2219254120. Epub 2023 Mar 27.
Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.
光遗传学是一种使用光在活细胞内对分子功能进行直接时空控制的技术。光的应用会诱导靶向蛋白发生构象变化，从而导致功能改变。光遗传学工具的应用之一是通过光感应结构域（LOV2）对蛋白质进行变构控制，从而可以直接、有效地控制蛋白质的功能。细胞成像的计算研究表明，光可以变构抑制信号蛋白 Vav2、ITSN 和 Rac1，但这种控制的结构和动态基础尚未通过实验阐明。在这里，我们使用 NMR 光谱学发现了细胞分裂控制蛋白 42（CDC42）变构控制的作用原理，CDC42 是一种参与细胞信号转导的小 GTPase。LOV2 和 Cdc42 在其功能中都利用了灵活性，分别在“暗”/“亮”或“活性”/“非活性”状态之间切换。通过将 Cdc42 和光敏素 1 LOV2 结构域结合到双开关融合 Cdc42Lov 中，应用光或模拟光吸收的 LOV2 突变可以变构抑制 Cdc42 的下游信号转导。在这个灵活的系统中，变构转导的流动和模式非常适合通过 NMR 进行观察。对 Cdc42Lov 的暗态和“亮”态的结构和动态特性进行密切监测，揭示了“亮”态诱导的变构扰动延伸到 Cdc42 的下游效应子结合位点。亮模拟物 I539E 的化学位移扰动具有不同的敏感区域，并且两个结构域相互耦合，导致双向的结构域间信号传递。从这种光变构设计中获得的见解将提高我们在未来设计中控制响应灵敏度的能力。
