Allosteric inactivation of an engineered optogenetic GTPase

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-sensitive LOV2 domain, which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically controlled 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 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 is 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. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity and both the domains are coupled together leading to bi-directional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs. Significance StatementControl of cell signaling activity in proteins by light is one of the primary goals of optogenetics. The hybrid light-receptor/cell-signaling protein Cdc42Lov was engineered recently as an optogenetic tool, employing a novel allosteric strategy that results in photoinhibition. In contrast to previous activation designs, the mechanism of inhibition of GTPase signaling activity in Cdc42 is only apparent at a detailed structural and dynamic level. NMR characterization of dark and mutationally "lit" forms reveals the allosteric interdomain perturbations, knowledge of which will enhance future applications of this design strategy.