Leveraging a Small‐Molecule Modification to Enable the Photoactivation of Rho GTPases

Control of both the timing and location of protein activation is essential for cell growth and movement. One of the key ways in which cells accomplish this control is through the use of molecular switches known as Rho GTPases. When loaded with GTP, these proteins are activated and can bind to effector proteins, which activate cellular responses (Figure 1 A). Subsequent hydrolysis of GTP to GDP returns the GTPase to the inactive GDP-bound state. The location of this molecular switch and the timing of its activation determine where and when cellular machinery is recruited to perform a specific biological function. Inappropriate activation of Rho GTPases alters this spatiotemporal control and is found to occur in many human diseases including cancer, hypertension, diabetes, asthma, pathogenic infection, and neurological disorders. Traditionally, GTPase functions in living cells have been studied by the overexpression or microinjection of mutant forms of the proteins. Constitutively active mutants cannot hydrolyze GTP, and thus remain perpetually in an active state. Dominant negative mutants inhibit the activation of endogenous GTPases, while fast-cycling mutants can cycle between active and inactive states independently of cellular regulation. The use of these mutants has yielded considerable insight into the roles of Rho GTPases in different cellular processes. However, this type of approach lacks the spatiotemporal control needed to dissect the individual roles of specific GTPases at different subcellular locations and times during the cell cycle. In an effort to overcome this limitation, we have developed a general approach to control Rho GTPase activation with light. Photoactivatable (“photocaged”) small molecules have been widely used to affect the rapid release of an active species at a defined location and time. 6] Proteins have been photocaged by unnatural amino acid mutagenesis approaches, 8] the use of phytochrome fusion proteins, and by direct covalent modification with photolabile small molecules. In vitro modification followed by loading into live cells has successfully been used to allow spatial and temporal control of cellular protein activity. Notable examples include protein kinase A (PKA), cofilin, and Smad2. Nonetheless, the modification, purification, and selective functional disruption of photocaged proteins remains challenging. To begin to address these challenges, we took advantage of the wealth of structural information available for the Rho GTPase family. Most Rho GTPases contain a cysteine residue within the nucleotide-binding pocket. We predicted that alkylation of this cysteine in the Rho GTPase Cdc42 would block the ability of GTP to bind and activate Cdc42 (Figure 1 B) and that subsequent irradiation with long-wave UV light would release this small molecule and restore Cdc42’s GTP-binding capacity (Figure 1 C). To test this idea, we attempted to alkylate cysteine 18 in a fast-cycling Cdc42 mutant (1–188, F28L/C188S). However, by using previously reported photocages such as 2-bromo-2(2-nitrophenyl)acetic acid 1 (Scheme 1), we found that: 1) we could not completely separate the modified protein from the unmodified protein, and 2) alkylation of this cysteine failed to completely prevent the ability of GTP to activate Cdc42 as determined by an effector pulldown assay. Because any contaminating uncaged protein or residual activity of the caged protein would compromise the spatial and temporal control afforded by photocaging, it is important that the caged protein is purified and inactive. As previously reported photocages failed to block Cdc42 activation, we synthesized novel photocages with additional steric bulk. However, these molecules either failed to block Cdc42 activation (for example, 2) or failed to alkylate cysFigure 1. Use of photolabile small molecules to control Rho GTPase activity. A) Exchange of GTP for GDP causes Rho GTPases to undergo a conformational change that allows them to interact with effector proteins and activate cellular responses. B) Alkylation of the endogenous Cys18 in the nucleotide-binding pocket of GTPases with a small-molecule photocage should prevent GTP binding and thus effector binding. C) Irradiation with UV light should remove the photocage; this would restore Cys18 and allow the GTPase to adopt the active conformation.