1XR0

Structural Basis of SNT PTB Domain Interactions with Distinct Neurotrophic Receptors


SOLUTION NMR
NMR Experiment
ExperimentTypeSample ContentsSolventIonic StrengthpHPressureTemperature (K)Spectrometer
1HNHASNT-1 PTB domain/hFGFR1 peptide complex (1:1) of ~0.5 mM in 100 mM phosphate buffer of pH 6.5, 5 mM DTT-d10, and 0.5 mM EDTA in H2O/2H2O (9/1) or 2H2OH2O/2H2O (9/1) or 100% 2H2O15 mM DTT-d10, and 0.5 mM EDTA00 mM phosphate buffer,6.51 atm303
23D_13C-separated_NOESYSNT-1 PTB domain/hFGFR1 peptide complex (1:1) of ~0.5 mM in 100 mM phosphate buffer of pH 6.5, 5 mM DTT-d10, and 0.5 mM EDTA in H2O/2H2O (9/1) or 2H2OH2O/2H2O (9/1) or 100% 2H2O15 mM DTT-d10, and 0.5 mM EDTA00 mM phosphate buffer,6.51 atm303
33D_15N-separated_NOESYSNT-1 PTB domain/hFGFR1 peptide complex (1:1) of ~0.5 mM in 100 mM phosphate buffer of pH 6.5, 5 mM DTT-d10, and 0.5 mM EDTA in H2O/2H2O (9/1) or 2H2OH2O/2H2O (9/1) or 100% 2H2O15 mM DTT-d10, and 0.5 mM EDTA00 mM phosphate buffer,6.51 atm303
42D NOESYSNT-1 PTB domain/hFGFR1 peptide complex (1:1) of ~0.5 mM in 100 mM phosphate buffer of pH 6.5, 5 mM DTT-d10, and 0.5 mM EDTA in H2O/2H2O (9/1) or 2H2OH2O/2H2O (9/1) or 100% 2H2O15 mM DTT-d10, and 0.5 mM EDTA00 mM phosphate buffer,6.51 atm303
NMR Spectrometer Information
SpectrometerManufacturerModelField Strength
1BrukerDRX500
2BrukerDRX600
NMR Refinement
MethodDetailsSoftware
Structures of the SNT-1 PTB domain in complex with the hFGFR1 peptide were calculated with a distance geometry and simulated annealing protocol by using the X-PLOR program ([4]). NOE distance and dihedral angle restraints were treated with a square-well potential of 50 kcal mol?1 ?2. A total of 2448 manually assigned NOE-derived distance restraints were obtained from the 15N- or 13C-edited NOESY data. Included in this figure are 251 intrapeptide and 258 intermolecular distance restraints. Additionally, 255 unambiguous and 52 ambiguous distance restraints were identified from the NOE data by using ARIA. The final structure calculations employed a total of 2755 NOE restraints obtained from the manual and the ARIA-assisted assignments, 2703 of which were unambiguously assigned NOE-derived distance restraints that comprise 1072 intraresidue, 466 sequential, 216 medium-range, and 949 long-range NOEs. In addition, 70 hydrogen-bond distance restraints for 35 hydrogen bonds and 19 -angle restraints were also used in the structure calculations. For the ensemble of the final 20 structures, no distance or torsional angle restraint was violated by more than 0.4 or 5, respectively. The distance-violation, dihedral-violation, and total energies were 74.4 1.7 kcal mol/1, 0.82 0.08 kcal mol/1, and 262.0 6.0 kcal mol/1, respectively. The Lennard-Jones potential, which was not used during any refinement stage, was 659.3 23.1 kcal mol/1 for the final structures. Ramachandran plot analysis by Procheck-NMR showed that in the final structures of the complex, 98.1% of the backbone geometries of the non-Gly and non-Pro residues in the complex (protein residues 18-116 and peptide residues (412-430) and nearly 100% in the secondary structure (protein residues 19-24, 35-40, 45-49, 52-57, 63-68, 71-76, 85-90, 94-107, and 111-115 and peptide residues 426-430) lie within energetically favorable or allowed regions.X-PLOR
NMR Ensemble Information
Conformer Selection Criteriastructures with the least restraint violations
Conformers Calculated Total Number100
Conformers Submitted Total Number1
Representative Model (fewest violations)
Additional NMR Experimental Information
DetailsNMR spectra were acquired at 30C on a Bruker DRX600 or DRX500 spectrometer. The backbone and side chain 1H, 13C, and 15N resonances of the protein were assigned using deuterium-decoupled triple-resonance experiments of HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, and (H)C(CO)NH-TOCSY ([34 and 30]) recorded by using uniformly 15N/13C-labeled and fractionally deuterated protein in complex with a nonisotopically labeled hFGFR1 peptide. The side chain assignments were completed using 3D HCCH-TOCSY ([7]) data collected from a uniformly 15N/13C labeled-protein/nonlabeled-peptide complex. NOE-derived distance restraints were obtained from 15N- or 13C-edited 3D NOESY spectra ([7]). -angle restraints were determined from 3JHN,H coupling constants measured in a 3D HNHA-J spectrum ([7]). Slowly exchanging amide protons were identified from a series of 2D 15N-HSQC spectra recorded after the H2O buffer was changed to 2H2O buffer. The peptide resonances were assigned using 13C/15N-filtered 2D NOESY and TOCSY spectra ([30]) collected from a 15N/13C labeled-protein/nonlabeled-peptide complex. The intermolecular NOEs used in defining the structure of the SNT-1 PTB domain/hFGFR1 complex were detected in 13C- or 15N-edited (F 1), 13C/15N-filtered (F 3) 3D NOESY spectra. All NMR spectra were processed with NMRPipe/NMRDraw ([8]) and analyzed using NMRView.
Computation: NMR Software
#ClassificationVersionSoftware NameAuthor
1structure solutionX-PLOR3.851Brunger
2refinementARIA1.1M. Nilges and S. O'Donoghue