Structural basis of pseudoGTPase-mediated protein-protein interactions

GTPases regulate various cellular processes through conformational changes triggered by GTP or GDP binding. Recently, pseudoGTPases, the catalytically inactive counterparts of GTPases, have been identified across species from bacteria to human, although their functions and mechanisms remain unexplored. In this study, we demonstrate that the N-terminal region of the assembly chaperone AAGAB is a type i pseudoGTPase using biochemistry and X-ray crystallography. Furthermore, we discovered that the AAGAB pseudoGTPase domain (psGD) interacts with the {sigma} subunits of AP1 and AP2 adaptor complexes, heterotetrameric complexes involved in clathrin-mediated membrane trafficking. AAGAB psGD engages the {sigma} subunits via a unique interface distinct from the conventional GTPase interacting regions. Further biochemical and cell-based assays confirmed the crucial role of the newly identified interface in binding and membrane trafficking. Collectively, our results establish AAGAB pseudoGTPase domain as a critical protein-protein interaction module. These findings offer new insight into the structural basis and molecular mechanisms of pseudoGTPases. HighlightsO_LIAAGAB N-terminal region is a pseudoGTPase C_LIO_LIAAGAB pseudoGTPase domain interacts with and stabilizes AP1 and AP2 {sigma} subunits C_LIO_LIThe crystal structure of AAGAB psGD:AP1{sigma}3 is the first reported psGD complex structure, revealing a unique interface independent of guanine nucleotide regulation C_LIO_LIThe psGD:AP1{sigma}3 structure offers mechanistic insights into {sigma} subunits stabilization and protection through adaptor complex assembly C_LI