Backbone Dynamics of an Oncogenic Mutant of Cdc42Hs Shows Increased Flexibility at the Nucleotide-Binding Site

• Published in: Biochemistry (Easton) Vol. 43; no. 31; pp. 9968 - 9977
• Main Authors: Adams, Paul D, Loh, Adrienne P, Oswald, Robert E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.08.2004
• Subjects: Amino Acid Sequence; Binding Sites - genetics; cdc42 GTP-Binding Protein - chemistry; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Deuterium Exchange Measurement; Guanine Nucleotides - chemistry; Guanine Nucleotides - metabolism; Humans; Leucine - genetics; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Oncogene Proteins - chemistry; Oncogene Proteins - genetics; Oncogene Proteins - metabolism; Phenylalanine - genetics; Point Mutation; Protein Binding - genetics; Protein Conformation; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0490901

Cdc42Hs, a member of the Ras superfamily of GTP-binding signal transduction proteins, binds guanine nucleotides, and acts as a molecular-timing switch in multiple signal transduction pathways. The structure of the wild-type protein has been solved (Feltham et al. (1997) Biochemistry 36, 8755−8766), and the backbone dynamics have been characterized by NMR spectroscopy (Loh et al. (1999) Biochemistry 38, 12547−12557). The F28L mutation of Cdc42Hs is characterized by an increased rate of cycling between the GTP and GDP-bound forms leading to cell transformation (Lin et al. (1997) Curr. Biol. 7, 794−797). Here, we describe the backbone dynamics of Cdc42Hs(F28L)-GDP using 1H-15N NMR measurements of T 1, T 1 ρ, and steady-state NOE at two magnetic field strengths. Residue-specific values of the generalized order parameters (S s 2 and S f 2), local correlation time (τe), and exchange rate (R ex) were obtained using the Lipari−Szabo formalism. Chemical-shift perturbation analysis suggested that very little structural change was evident outside of the nucleotide-binding site. However, residues comprising the nucleotide-binding site, as well as the nucleotide itself, exhibit increased dynamics over a wide range of time scales in Cdc42Hs(F28L) relative to the wild type. In addition to changes in dynamics measured by relaxation methods, hydrogen−deuterium exchange indicated a substantial disruption of the hydrogen-bonding network within the nucleotide-binding site. Thus, local dynamic changes introduced by a single-point mutation can affect important aspects of signaling processes without disrupting the conformation of the whole protein.