SOLUTION STRUCTURE OF THE FAT DOMAIN OF FOCAL ADHESION KINASE
NMR Solution Structure of the Focal Adhesion Targeting Domain of Focal Adhesion Kinase in Complex with a Paxillin LD Peptide: EVIDENCE FOR A TWO-SITE BINDING MODEL.
RSNDKVYENVTGLVKAVIEMSSKIQPAPPEEYVPMVKEVGLALRTLLATVDESLPVLPASTHREIEMAQKLLNSDLAELI NKMKLAQQYVMTSLQQEYKKQMLTAAHALAVDAKNLLDVIDQARLKMISQSRPH
HELIX Determination method: Author determined
Aromatic sidechain assignments were obtained from 2D (HB)CB(CGCDCE)HE and 2D (HB)CB(CGCD)HD experiments. 1H-15N residual dipolar coupling values were measured using 7.5 mg/ml Pf1 phage as aligning medium
The structural restraints included NOE, hydrogen bond, dihedral angle, and RDC restraints. The NOE distance restraints were derived from a 3D simultaneous 13C/15N edited NOESY peak lists with 75 ms mixing time. The peak lists were generated automatically by NMRView and edited manually to remove any obvious water and apodization artifacts. The peak lists were unassigned and uncalibrated with respect to distance. To calibrate the NOE distances, the rotational correlation time of the complex was set to 10 ns and was based on values obtained on proteins of this size at similar temperature. The default parameters for ARIA produced structures with poor convergence. Several parameters were optimized, including the ambiguous cutoff (rho), the violation tolerance (vtol), and maximum number of ambiguities per peak (maxn), to provide better noise discrimination. The following scheme led to the best convergence over 9 iterations: rho = (0.95, 0.95, 0.95, 0.94, 0.93, 0.92, 0.91, 0.90, 0 80), vtol = (5.0, 2.0, 1.0, 0.5, 0.25, 1.0, 0.1, 0.1, 0.1), maxn = 5 for all iterations. An ensemble of 25 calculated NMR structures of the avian FAT domain was selected for further analysis.
The program CNS version 1.1 with ARIA version 1.2 module was used to calculate the structures of the avian FAT domain.
0.6mM 13C,15N labeled FAT with 4.8mM LD2 peptide, 25mM TRIS-MALEATE, 0.1% NAN3, 1.0uM PPACK, 0.5mg/mL PEFABLOC, 90% H2O, 10% D2O
20mM deuterated Tris-maleate, 50mM NaCl, 0.1mM EDTA, 0.1% NaN3, and 10% D2O, pH 6.0
FOCAL ADHESION KINASE, FAK, FOCAL ADHENSION TARGETING DOMAIN, FAT, HELIX BUNDLE, TRANSFERASE