Backbone Dynamics of Inactive, Active, and Effector-Bound Cdc42Hs from Measurements of15N Relaxation Parameters at Multiple Field Strengths

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Abstract
Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs and found that the regions involved in effector (Switch I) and regulator (Switch II) actions are partially disordered [Feltham, J. L., et al. (1997) Biochemistry36, 8755−8766]. Recently, we used a 46-amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes the β2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemistry37, 14030−14037]. Here we describe the backbone dynamics of three constructs of [15N]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using 15N−1H NMR measurements of T1, T1ρ, and the steady-state NOE at three magnetic field strengths. Residue-specific values of the generalized order parameters (Ss2 and Sf2), local correlation time (τe), and exchange rate (Rex) were obtained using the Lipari−Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) motions on a nanosecond time scale, whereas those in Switch II experience low-amplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhibits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs−GMPPCP and Cdc42Hs−PBD46. Overall, significant flexibility was observed mainly in the regions of Cdc42Hs that are involved in protein−protein interactions (Switch I, Switch II, and Insert), and flexibility was reduced upon interaction with a protein ligand. These results suggest that protein flexibility is important for high-affinity binding interactions.