Backbone dynamics of an oncogenic mutant of Cdc42Hs shows increased flexibility at the nucleotide-binding site.
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 T1, T1rho, and steady-state NOE at two magnetic field strengths. Residue-specific values of the generalized order parameters (Ss2 and Sf2), local correlation time (tau(e)), 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.
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Paul D
PD
Adams
Adrienne P
AP
Loh
Robert E
RE
Oswald
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D041961:Deuterium Exchange Measurement; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006150:Guanine Nucleotides; D015513:Oncogene Proteins; D010649:Phenylalanine; C081345:Cdc42Hs-associated kinase; D011505:Protein-Tyrosine Kinases; D020764:cdc42 GTP-Binding Protein; D007930:Leucine