A complete allosteric map of a GTPase switch in its native cellular network

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. [Display omitted] •Comprehensive mutation of the conserved GTPase switch Gsp1/Ran in its native network•Allosteric sites are prevalent and widely distributed throughout the GTPase structure•Modulation of GTPase switch kinetics is a shared mechanism of diverse regulatory sites•Comparative analysis reveals conserved and subfamily-specific regulatory sites Allostery is essential to protein regulation in cellular networks but to date no comprehensive allosteric map exists for any protein. We determine a map for allosteric regulation of a GTPase in its native biological network. We discover 20 allosteric sites, opening avenues for targeting GTPases controlling many essential biological processes.