Computational protocol: The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4

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Protocol publication

[…] Chemical shifts for LMO4LIM2•DEAF1404–418, in 20 mM sodium acetate at pH 5.0, 35 mM NaCl, 0.5 mM TCEP-HCl, 34 µg mL–1 chloramphenicol and Complete EDTA-free protease inhibitor (one tablet per 50 mL), were assigned using standard triple resonance NMR experiments as described in . Distance restraints were obtained from 1H-1H 2D NOESY, 15N-edited NOESY and 13C-edited NOESY spectra, all with mixing times of 150 ms. NOE peaks were manually picked, checked and corrected where necessary. The ensemble of structures for LMO4LIM2•DEAF1404–418 was calculated using ARIA 1.2 implemented in CNS 1.21 . Default parameters were used except where stated. The number of molecular dynamics steps was: initial stage, 40000; refinement stage, 8000; first cooling step, 40000; and second cooling step, 8000. The upper limit for NOE distance estimates was increased by 0.15 Å from the default value. A mixing time of 150 ms and a rotational correlation time of 5.16 ns were used to set relaxation matrix parameters. A zinc patch was included to define zinc co-ordination geometry () based on the coordinates of the LIM2 domains from LMO4 and LMO2 in the LMOLIM1+2•LDB1LID (PDB IDs: 1RUT and 2XJY, respectively. ). In the first iteration 200 structures were calculated, with 20 structures calculated for each of the 7 intermediate iterations, and 600 structures in the final iteration. The 50 lowest energy structures from the final iteration were further refined in a shell of water using the standard ARIA protocol. Longitudinal (T 1), transverse (T 2) and heteronuclear NOE relaxation experiments were performed on 600 µM 15N-labelled LMO4LIM2•DEAF1404–418 using the Bruker pulse programs hsqct1etf3gpsi3d, hsqct2etf3gpsi3d and hsqcnoef3gpsi3d, respectively. The relaxation delays used for measuring 15N-T 1 time constants were 0.1, 0.15, 0.2, 0.3, 1, 1.4, 1.5 and 2.2 s and those for measuring T 2 time constants were 17, 34, 51, 68, 85, 102, 136, 153, 170, 221 and 255 ms. The same relaxation experiments were recorded at 800 MHz except that the T 2 relaxation delays at 800 MHz were 16, 32, 48, 64, 80, 96, 128, 144, 160, 208 and 240 ms. Lipari-Szabo ordered parameters (S2) were calculated using the model-free module (fully automated mode) in relax , .Recycle delays of 4 s were used in these experiments. Integrated peaks were fitted to two-parameter exponentials using the relaxation analysis module in SPARKY . 1H-15N heteronuclear NOEs were calculated by taking the ratio of cross-peak intensities with and without proton saturation during relaxation delays. One-dimensional 1H and two-dimensional 15N-HSQC spectra of L4-DEAF1 were performed in 20 mM sodium acetate at pH 5.0 and 35 mM NaCl. Images of structures were generated using PyMol, simple homology models were generated using SWISS-MODEL or mutation of residues in PyMol, and the surface area of the LMO4-DEAF1 interface was calculated using PISA . […]

Pipeline specifications

Software tools Sparky, PyMOL, SWISS-MODEL
Applications NMR-based proteomics analysis, Protein structure analysis
Diseases Breast Neoplasms