Concerted motion of a protein-peptide complex: backbone dynamics studies of an (15)N-labeled peptide derived from P(21)-activated kinase bound to Cdc42Hs.GMPPCP.
Cdc42Hs is a member of the Ras superfamily of GTPases which, when active, initiates a cascade beginning with the activation of several kinases, including P(21)-activated kinase (PAK). We previously determined the structure of a complex between a 46 amino acid fragment peptide derived from the PAK binding domain (PBD46) and Cdc42Hs.GMPPCP (Gizachew, D., Guo, W., Chohan, K. K., Sutcliffe, M. J., and Oswald, R. E. (2000) Biochemistry 39, 3963-3971). Previous studies (Loh, A. P., Guo, W., Nicholson, L. K., and Oswald, R. E. (1999) Biochemistry 38, 12547-12557) suggest that the regions of Cdc42Hs that bind effectors and regulators have distinct dynamic properties from the remainder of the protein. Here, we describe the backbone dynamics of PBD46 bound to Cdc42Hs.GMPPCP. T(1), T(2), T(1)(rho), and steady-state nuclear Overhauser effects were measured at 500 and 600 MHz. An extension of the Lipari-Szabo model-free analysis was used to determine the order parameters (S(2)) and local correlation times (tau(e)) of the N-H bond vectors within PBD46. 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 of PAK. Different backbone 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 part of PBD46 that interacts with Switch I of Cdc42Hs exhibits greater mobility. Thus, PBD46 and Cdc42Hs form a tight complex that exhibits concerted dynamics.
Gizachew
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Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.
Oswald
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D000595:Amino Acid Sequence; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein; D054462:p21-Activated Kinases
D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D051316:Wiskott-Aldrich Syndrome Protein; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; C081345:Cdc42Hs-associated kinase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases