"Self-gapping" by a C-terminal domain arginine finger regulates GTP hydrolysis in bacterial zinc metallochaperones.

The cellular response to transition metal scarcity is multifaceted and complex. Members of the Cluster of Orthologous Groups 0523 (COG0523) superfamily are proposed to chaperone a bound metal to activate an apoenzyme client and are thus candidate metallochaperones. COG0523 enzymes are GTPases that harbor a conserved Ras-like GTP-binding and hydrolysis domain (G-domain) and a C-terminal domain (CTD) of unknown function connected by a flexible linker. AlphaFold3 modeling posits an "open" GTPase-inactive and "closed" GTPase-active conformation where the GTP and switch 1 (G2) loop are buried at the interface of the two domains. We show here that the CTD functions as a GTP-hydrolysis activation protein (GAP) domain that stimulates GTP hydrolysis by the tethered G-domain. This "self-gapping" activity requires an invariant RxKG sequence in the β2-strand of the CTD in two distantly related bacterial COG0523s from Significance Statement: The cellular response to nutrient transition metal limitation is evolutionarily conserved in all kingdoms, providing protection from the loss of these essential inorganic cofactors that power much of metabolism. An important part of this response is the increased cell abundance of members of the enigmatic and ubiquitous Cluster of Orthologous Groups 0523 (COG0523) superfamily. In bacterial pathogens, these enzymes are often associated with the low-zinc adaptive response to host-mediated nutritional immunity, where the host deploys transition metal chelation as an innate immune response to infections. In this work, we provide new mechanistic insights into COG0523 function with the discovery of "self-gapping" by the C-terminal domain of a two-domain G-protein architecture, placed into the context of a metallochaperone model.