Concerted motion of a protein-peptide complex: Backbone dynamics studies of an N-15-labeled peptide derived from P-21-activated kinase bound to Cdc42Hs center dot GMPPCP
D. Gizachew et Re. Oswald, Concerted motion of a protein-peptide complex: Backbone dynamics studies of an N-15-labeled peptide derived from P-21-activated kinase bound to Cdc42Hs center dot GMPPCP, BIOCHEM, 40(48), 2001, pp. 14368-14375
Cdc42Hs is a member of the Ras superfamily of GTPases which, when active, i
nitiates a cascade beginning, with the activation of several kinases, inclu
ding P-21-activated kinase (PAK). We previously determined the structure of
a complex between a 46 amino acid fragment peptide derived from the PAK bi
nding, domain (PBD46) and Cdc42Hs(.)GMPPCP (Gizachew, D., Guo, W., Chohan,
K. K.. Sutcliffe. M. J., and Oswald, R. E. (2000) Biochemistry 39, 3963-397
1). Previous Studies (Loh, A. P.. Guo. W.. Nicholson, L. K., and Oswald, R.
E. (1999) Biocheinistry, 38, 12547-12557) suo-est that the regions of Cdc4
2Hs that bind effectors and regulators have distinct dynamic properties fro
m the remainder of the protein. Here. we describe the backbone dynamics of
PBD46 bound to Cdc42Hs-GMPPCP. T-1. T-2. T-1p, and steady-state nuclear Ove
rhauser effects were measured at 500 and 600 MHz. An extension of the Lipar
i-Szabo model-free analysis was used to determine the order parameters (S-2
) and local correlation times (tau (c)) of the N-H bond vectors within PBD4
6. Both Cdc42Hs and PBD46 exhibit increased mobility in the free versus the
bound state, suggesting that protein flexibility may be required for high-
affinity PBD46 binding and, presumably, the activation or PAK. Different ba
ckbone dynamics were observed in different regions of the peptide. The beta
-strand region of bound PBD46, which makes contacts with beta2 of Cdc42Hs.
exhibits low mobility on the pico- to nanosecond timescale. However, the p
art of PBD46 that interacts with Switch I of Cdc42Hs exhibits greater mobil
ity. Thus, PBD46 and Cdc42Hs form a tight complex that exhibits concerted d
ynamics.