Computational protocol: Structural insights into leishmanolysins encoded on chromosome 10 of Leishmania (Viannia) braziliensis

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Protocol publication

[…] Sequence analysis - We retrieved the L. (V.) braziliensis sequences corresponding to the metalloprotease family from the GeneDB (). Sequence similarity searches were performed using the BLASTp algorithm, and a multiple sequence alignment of the obtained sequences was carried out using T-Coffee Multiple Sequence Alignment Server (). Comparative modeling - Candidate template structures for subsequent comparative modeling were taken from the PDB database (). Three-dimensional models were generated with the Swiss-Model server () employing as a template the high-resolution structure of L. major leishmanolysin (PDBid: 1LML) (), maintaining all input parameters as default. Three homology models were created for each submitted sequence and the structure displaying the highest QMEAN score () was utilised for further analysis. The stereochemical quality of the models was evaluated with the structure analysis and verification server (SAVES) (). Loop optimisations were performed with the KoBaMIN server (). MD simulations - MD simulations were carried out using AMBER 14.0 in conjunction with the ff14SB force field (, ). Electrostatic interactions were treated using the particle mesh ewald (PME) algorithm with a cut-off of 10 Å. Each system was simulated under periodic boundary conditions in a cubic box filled with TIP3P water molecules (). To properly describe the coordination of the zinc ion within the active site, the protonation states of surrounding histidine residues were manually assigned: they were selected as Nd neutral tautomers. All systems were neutralised by adding counterions.Subsequently, a two-step energy minimisation procedure was performed: (i) 2000 steps [1000 steepest descent (SD) + 1000 conjugate-gradient (CG)] with all heavy atoms harmonically restrained with a force constant of 5 kcal mol-1 Å-2; (ii) 5000 steps (2500 SD + 2500 CG) without position restraints. Next, initial atomic velocities were assigned using a Maxwell-Boltzmann distribution corresponding to an initial temperature of 20 K and the systems were gradually heated from 20 K to 310 K over one nanosecond utilising the Langevin thermostat. During this stage, all heavy atoms were harmonically restrained with a force constant of 10 kcal mol-1 Å-2. Systems were subsequently equilibrated during nine successive 100 ps equilibration simulations where position restraints approached zero progressively. After this period, the systems were simulated with no restraints at 310 K for 50 ns in the Gibbs ensemble with a pressure of 1 atm. Atomic coordinates and energies were recorded every 25 ps. The simulation trajectories were analysed using GROMACS package tools (). Electrostatic potential analysis and geometrical data from the active site - We firstly performed a clustering analysis of the simulation trajectories with the module gmx cluster of the GROMACS package. In this step, we used the gromos algorithm () to identify conformational populations with an 1Å RMSD cut-off. The central structure corresponding to the most populated cluster in each simulation was submitted for subsequent calculations.The electrostatic potential analysis was conducted with the APBS program (). The AmberFF charge and radii parameters were assigned using the PDB2PQR server (). Subsequently, these structures were submitted to the 3V server () to explore structural features of the active site (volume, surface area, sphericity, and effective radius). The characterization of the active site binding cavities was performed with KVFinder using default parameters (. […]

Pipeline specifications

Software tools BLASTP, T-Coffee, SWISS-MODEL, QMEAN, KoBaMIN, AMBER, GROMACS, PDB2PQR, KVFinder
Databases GeneDB
Application Protein structure analysis
Organisms Leishmania braziliensis
Diseases Infection