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.
Adams
Paul D
PD
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Loh
Adrienne P
AP
Oswald
Robert E
RE
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