Control of Guanine Exchange Factor Activity Using De Novo Designed Protein Inhibitors

Guanine Exchange Factors (GEF) of the Dbl family are the main activators of RhoA GTPases. GEF and GTPase activity is tightly regulated at the subcellular level with fast kinetics. Therefore, to fully understand the function of Dbl GEFs requires their study in living cells. Towards developing molecular tools that reversibly and rapidly modulate the activity of endogenous GEFs in living cells, here we developed a general platform for engineering inhibitors against members of the Dbl family of GEFs using generative protein design. Engineered proteins showed high affinity and remarkable specificity for the target GEFs and modulated GEF activity both in vitro and in cells. In a proof-of-principle example, a GEF inhibitor was coupled to a light-activated module, enabling the optogenetic control of its activity in cells. These findings show that generative protein design can create modulators of intracellular signaling and broaden the range of tools available for biological research.