Computational protocol: NMR Structure of Integrin α4 Cytosolic Tail and Its Interactions with Paxillin

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Protocol publication

[…] All NMR experiments were recorded on a Bruker DRX 600-MHz instrument equipped with an actively shielded cryo-probe. 10% Deuterium oxide and 2 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was added to all NMR samples. Chemical shifts were referenced to DSS. 2D TOCSY (mixing time: 50 ms) and 2D NOESY (mixing time: 200 ms) spectra were recorded for 0.5 mM of α4 CT dissolved in water, pH 5.6 at 278 K. Raw NMR data were processed using TOPSPIN 2.1 and analyzed with SPARKY. 15N-1H HSQC spectra of α4 CT and PaxLD2-LD4 were assigned by triple resonance HNCACB and CBCA(CO)NH experiments. Triple resonance NMR experiments were carried out using doubly labeled (15N/13C) samples of α4 CT and PaxLD2-LD4 dissolved in 10 mM sodium phosphate buffer, pH 5.6, at 298K. For interactions studies, 15N-1H HSQC spectra of either 15N-labeled α4 CT (100 µM) or PaxLD2-LD4 (200 µM) were obtained in the presence of unlabeled binding partners at molar ratio of 1∶1 and 1∶2 in 10 mM sodium phosphate buffer, pH 6.5, 298 K. [...] NOE intensities of α4 CT NOESY spectra were qualitatively categorized into strong, medium and weak and translated to the upper bound distance limit of 2.5 Å, 3.5 Å and 5.0 Å respectively. These distance constraints were used for structure calculations using CYANA (Combined assignment and dynamics for NMR applications) 2.1 . For structure calculation backbone dihedral angle (Φ) values were restricted to −30° to 120° to limit the conformational search. Of the 100 structures, 20 lowest energy structures were selected for evaluation and analyses. PROCHECK-NMR was employed to evaluate the stereochemical quality of the structural ensemble and figures were prepared using PyMOL, MOLMOL, Discovery Studio Visualizer 2.0 and Insight II. Docking of α4 CT with LD peptides of paxillin was performed using Insight II software. Helical structures of LD3 and LD4 peptide fragments were constructed for docking with α4 CT. Several round of docking exercises were conducted to achieve optimal sidechain-sidechain packing interactions. The model complex was further energy minimized using discover force field to relieve short inter-atomic contacts. […]

Pipeline specifications

Software tools Sparky, PROCHECK, PyMOL, MOLMOL, Discovery Studio® Visualizer
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Homo sapiens
Chemicals Hydrogen