Computational protocol: Solution Structure of a Phytocystatin from Ananas comosus and Its Molecular Interaction with Papain

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Protocol publication

[…] Resonance assignments of free AcCYS were obtained using a series of double- and triple-resonance NMR experiments and have been reported in our previous study . To assign NOE-based distance restraints, 3D 13C-separated and 15N-separated NOESY-HSQC spectra were recorded on uniformly 13C and 15N-labeled samples respectively. The NOE crosspeaks were picked and quantified by peak-picking algorithm in Aurelia. The NOE peak intensities were converted into upper distance bounds using CALIB module in the torsional angle dynamics program CYANA . The backbone dihedral angles (φ and ψ) restraints were derived by chemical shifts of 1Hα, 13Cα, 13Cβ, 13CO, and 15N nuclei using the program TALOS . Hydrogen bond restraints were assigned between slowly exchanging amide protons and their respective carbonyl acceptors deduced from the NOE data in combination with the secondary structure information predicted from CSI . Initial structures were generated by the automated module, CANDID/NOEASSIGN in CYANA. These NOE assignments were carefully confirmed and erroneous ones corrected through examination of spectra. Additional NOE were then added manually before recalculation of structures by CYANA. The final 20 conformers with the lowest target function values were selected and further refined by the restrained simulated annealing and energy minimization algorithms in CNS 1.3 , . Graphical visualization and analyses of the structures were carried out with the programs MOLMOL and PyMOL (DeLano Scientific). The geometric and stereo-chemical quality of the ensemble of structures was validated by PROCHECK-NMR . [...] Bioinformatics analysis of AcCYS with other cystatin sequences from different families was performed with CLC workbench (CLC bio). Multiple sequence alignments were performed using CLUSTALW algorithm . Prediction of signal peptide was performed using Target P 1.1 server . The solved structure of AcCYS was analyzed and classified by CATH and DALI algorithms. [...] Molecular modeling of AcCYS in association with papain and their docking and restrained molecular dynamic simulation were performed using Discovery Studio 2.1 platform (Accelrys, San Diego, CA). The initial structure model of AcCYS/papain complex was generated from the taro cystatin/papain crystal structure (PDB ID: 3IMA) with the taro cystatin replaced by our NMR AcCYS structure (PDB ID: 2L4V). This complex structure was then energy minimized with CHARMM force field . Docking simulations of AcCYS to papain were performed by use of rigid body ZDOCK 2.1 . The binding site of AcCYS and papain was defined as those residues with significant chemical shifts perturbation upon complex formation and in close contact (≤5 Å) with each other in our initial structure model. The orientation of AcCYS was obtained with 6° rotational sampling, and 54000 predictions were generated. The structure with the lowest ZRank score was selected and subsequently subject to energy minimization. The complex structure of AcCYS/papain obtained from ZDock was then further simulated with restrained molecular dynamics using a cascade protocol in Discovery Studio 2.1. The procedures in the restrained molecular dynamics simulation included minimization with steepest descent and conjugate gradient methods, followed by heating and equilibration dynamics under 300 K, and finished with production dynamics. The distance restraints used in the molecular dynamics simulation include hydrogen bonds derived from H/D hydrogen exchange and CSI data, intramolecular restraints of hydrophobic clusters in AcCYS, and intermolecular restraints derived from the dock model. The distance-dependent dielectric constant of the solvent was used in the molecular dynamics simulation. The lowest dielectric constant was 1 and the solvent dielectric constant was 80. The conformation of the AcCYS/papain complex with the lowest potential energy in the production dynamics was selected to present the complex structure of AcCYS/papain. […]

Pipeline specifications

Software tools CHARMM, ZDOCK
Application Protein interaction analysis
Organisms Ananas comosus