Computational protocol: Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study

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Protocol publication

[…] The protein sequence of SAAT was retrieved from NCBI protein sequence database (GeneBank accession number: AF193789). BLAST [] (Basic Local Alignment Search Tool) and PSIPRED (Protein Structure Prediction Server) [] were used to select the available 3D protein structures with the closest homology in the Brookhaven Protein Data Bank (PDB) []. The chosen template was the crystal structure of protein vinorine synthase (PDB ID: 2BGH) from Rauvolfia serpentina that shares 33% of sequence identity with SAAT, including important conserved motifs from BAHD superfamily []. The crystal structure of the template is complete and has a resolution of 2.6 Å. Despite the low sequence identity between members of BAHD family, the selected crystal structure shows a high similarity with several CoA-dependent acyltransferases. Moreover, it has been established that a structural identity over 30% is an acceptable threshold that guarantees a successful homology modeling process []. A pair-wise sequence alignment, between SAAT and the template, was obtained by using CLUSTAL W software [], in order to build the initial model. The homology model of SAAT structure was generated by using MODELLER 9v2 software (www.salilab.org/modeller) []. Fifty models were generated; from which five models were selected according to the DOPE (Discrete Optimized Protein Energy) [] score and their root mean squared deviation (RMSD) with respect to the trace of Cα atoms of the reference crystal structure. A loop optimization protocol was used to improve the quality of the five models. Finally, in order to select the best model among the 5 remaining, we evaluated the Phi and Psi angles using PROCHEK []. The model with the best stereochemistry was chosen for further analysis.In order to refine the structural model and to get the best conformation of SAAT structure, its conformational space was studied by means of molecular dynamics simulations (MDS) using Nanoescale Molecular Dynamics (NAMD v2.6) software and the Chemistry at HARvard Molecular Modeling (CHARMM27) force field for lipids and proteins [], along with the TIP3P water model []. The tautomeric states of Histidine residues in the model were assigned according to their local environment through the PROPKA [] utility. After an initial minimization, the system was subjected to a short MDS in order to remove wrong atomic contacts and to fill empty pockets, and finally to stabilize the energy of the model. To do so, 65000 steps were run using the conjugated-gradient method. All backbone atoms were restrained using a harmonic force constant of 0.5 kcal mol-1Å-2 and the loops were allowed to move during relaxation. All MDS were done using a timestep of 1 femtosecond (fs) with a 12Å spherical cutoff for non-bonding interactions and a switching function of 9Å for the van der Waals term. The structure obtained was embedded, using periodic boundary conditions (PBC), into a box of 78 x 86 x 92 Å3 contained in a 150 mM NaCl solution at 300 K and the isobaric-isothermal ensemble (NPT). Under these conditions, 2 ns of MDS were performed, using NAMD v2.6 software. Particle Mesh Ewald (PME) was employed to calculate long-range electrostatic interactions. Finally, the accuracy of the model was evaluated again through ANOLEA [], [] and PROCHECK []. […]

Pipeline specifications

Software tools BLASTN, PSIPRED, Clustal W, MODELLER, NAMD, PROPKA, PROCHECK
Databases NCBI Protein
Application Protein structure analysis
Diseases Genetic Diseases, Inborn
Chemicals Acyl Coenzyme A, Ethanol, Alcohols, Carbon, Esters, Oxygen