Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participati ng in nucleocytoplasmic transport, have been identified in different specie s ranging from yeast to man. All RanGAPs are characterized by a conserved d omain consisting of eight leucine-rich repeats (LRRs) interrupted at two po sitions by so-called separating regions, the latter being unique for RanGAP s within the family of LRR proteins. The cytosolic RanGAP activity is essen tial for the Ran GTPase cycle which in turn provides directionality in nucl eocytoplasmic transport, but the structural basis for the interaction betwe en Ran and its GAP has not been elucidated. In order to gain a better under standing of this interaction we generated a number of mutant RanGAPs carryi ng amino acid substitutions in the LRR domain and analysed their complex fo rmation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficien t Ran binding and GAP activity. These separating regions contain three cons erved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and repl acement of the third conserved arginine (Arg91 in human RanGAP) severely in terferes not only with GAP activity but also with Ran binding. This indicat es that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalyti c arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.