The functions of some conserved motifs of Ran and Ran binding domain 1 and 2 of RanBP2 were characterized by the kinetics and equilibrium of spectroscopy using analogues of guanine nucleotides, Isothermal Titration Calorimetry (ITC) and structural
analysis (crystallization and NMR) approaches. The results suggest 1) The C-terminal DEDDDL motif destabilizes GTP complexation with Ran. This tail of Ran is important for binding to BD1. 2) The N-terminal fragment of BD1 contributes largely to the
recognition of Ran in its GTP form. 3) The conserved 57WKER
motif of BD1 stabilizes GTP binding on Ran by interactions with the effector loop of Ran. 4) BD1 binds to Ran in its GDP form with a weak affinity of 50 μM. It promotes the dissociation rate of GDP from the Ran*GDP complex putatively by deformation of
of Ran in the GDP form. 5) NMR studies reveal that there is an extra 5th-β strand in the core domain of BD2 in solution, which make BD2 a perfect canonical PH domain.