Backbone dynamics of inactive, active, and effector-bound Cdc42Hs from measurements of N-15 relaxation parameters at multiple field strengths

Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p2 1-activated kinase (PAK). We have previously determined the structure of Cd c42Hs and found that the regions involved in effector (Switch I) and regula tor (Switch II) actions are partially disordered [Feltham, J. L., et al. (1 997) Biochemistry 36, 8755-8766]. Recently, we used a 46-amino acid fragmen t of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes t he beta 2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemi stry 37, 14030-14037]. Here we describe the backbone dynamics of three cons tructs of [N-15]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using N-15-H-1 NMR measurements of T-1, T-1 rho, and the steady-state NOE at thre e magnetic field strengths. Residue-specific values of the generalized orde r parameters (S-s(2) and S-f(2)), local correlation time (tau(e)), and exch ange rate (R-ex) were obtained using the Lipari-Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) moti ons on a nanosecond time scale, whereas those in Switch II experience low-a mplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhi bits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs-GMPPCP and Cdc42Hs-PBD46. Overall, significant flexi bility was observed mainly in the regions of Cdc42Hs that are involved in p rotein-protein interactions (Switch I, Switch Il, and Insert), and flexibil ity was reduced upon interaction with a protein ligand. These results sugge st that protein flexibility is important for high-affinity binding interact ions.