Computational protocol: Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study

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Protocol publication

[…] Electrostatic potential surfaces were computed with the APBS program . The charges and radius of all atoms were calculated using PDB2PQR software . The linearized traditional Poisson–Boltzmann equation was solved with APBS using a cubic spline charge discretization and with dielectric constants of 2.0 for the solute and 78 for the solvent at 300 K. Electrostatic potential is represented by a positive and a negative isosurface at ± 10 KbT/e (Kb, Boltzmann's constant; T, temperature and e, charge of an electron).All molecular dynamics simulations were performed using the NAMD 2.10 software in the isobaric–isothermal ensemble. The pressure (1 atm) and temperature (300 K) were kept constant using Langevin dynamics and Nosé–Hoover Langevin piston , . All protein atoms were described by the CHARMM27 force field while the aminoglycosides were parameterized using the paratool plugin implemented in VMD by assigning both the atomic partial charges computed with Gaussian (by fitting the electrostatic potential surface, ESP) and atom types from CHARMM27. The crystal structures containing either kanamycin A (PDB: 4DFB) or tobramycin (PDB: 3SG8) were immersed in a rectangular water box (TIP3 model) with a 12 Å thickness and neutralized with three or four Na+ ions. As the crystal structure of APH(2″)-IVa·sisomicin was not fully solved or unsatisfactory, the complex was obtained by docking (GOLD 5.2 program, CCDC Software Limited) into the chain B of the 4DFB crystal structure. Kanamycin was first removed and used as a center for the search of binding modes (docking poses) of sisomicin by applying 50 runs of genetic algorithms. Solutions were classified according to their scores using the goldscore function. This complex was immersed in a water box as the two other aminoglycosides. The solvated systems were replicated in each direction using periodic boundary conditions. The short-range Lennard–Jones potential was smoothly truncated from 10 to 12 Å and the PME (Particle Mesh Ewald) algorithm was used to calculate long-range electrostatics with a grid spacing of 1 Å. The potential energy of the molecular systems was minimized for 50,000 steps of conjugate gradient (time step of 1 fs). After a gradual heating from 0 to 300 K, the two systems were further equilibrated for 100,000 steps. To explore the “unbinding” of the aminoglycoside from the APH binding site, a reaction coordinate was defined as the distance separating the center of mass of each entity (antibiotic and protein). A biased force was applied using Colvars module of NAMD in order to estimate the free energy change along the reaction coordinates using the ABF method . The distance separating the two centers of mass was initially 7.5 Å and forced to reach 27.5 Å. A width of 0.1 Å was selected using four short window (5 Å) simulations (6 ns each) and the final potential of mean force (PMF) was reconstructed from these separate windows. Simulations were carried out on IBM blade cluster and trajectories were analyzed using VMD. [...] Crystals of binary complexes of APH(2″)-IVa were grown at 18 °C using similar conditions to those previously described by Toth et al. . Co-crystallization of the enzyme with either sisomicin or G418 (Geneticin) was carried out by mixing 1 μL of protein (6 mg/mL− 1) and aminoglycoside (2.5 mM) in 50 mM Hepes pH 7.5, 10 mM MgCl2 with 1 μL of reservoir solution composed of 12% PEG3350 (w/v), 50–75 mM ammonium citrate at pH 7.4 to 7.9.Crystals of binary complex APH(2″)-IVa·sisomicin were grown by the hanging drop vapor diffusion method over a reservoir of 500 μL. After 2–3 days, crystals with approximate dimensions of 300 × 100 × 50 μm were observed. They were briefly immersed in the reservoir solution supplemented with 15% of glycerol as cryoprotectant before being flash-cooled in liquid nitrogen. Data were collected under cryogenic conditions on a Pilatus 6M-F detector at ESRF (Grenoble, France) on beamline ID23-1. Crystals of binary complex APH(2″)-IVa·G418 were grown by the sitting drop vapor diffusion method over a reservoir of 40 μL. The approximate size of crystals was 100 × 50 × 50 μm. Data collection was carried out in situ as described by Gelin et al. in a 96-well CrystalQuickTM X plate (Greiner BioOne) on beamline BM30A-FIP at the ESRF, equipped with an ADSC Q315r CCD detector. Only the density obtained in chain A was sufficient enough to build G418.Both structures were refined by molecular replacement using chain A of the homologous APH(2″)-IVa·ADP complex (PDB: 4N57) after removal of the ligand as search model. Data were processed and scaled with XDS and SCALA . The atomic models were rebuilt in Coot and refined using Refmac with 10 sub-segments per protein chain of torsion–libration–screw from the TLSMD server . G418 and sisomicin restraints were generated with the PRODRG2 server . The 2Fo − Fc omit maps were calculated with AutoBuild from the PHENIX package after omitting the aminoglycoside molecules present in the asymmetric unit. Data collection and refinement statistics are shown in and the structures were deposited in the protein databank (PDB: 5C4K and PDB: 5C4L). The B-factor values for G418 were large which may be explained by the high flexibility of the aminoglycoside in the binding site.The structures were analyzed using the PyMOL Molecular Graphics System (version 1.3, Schrödinger, LLC). The rmsd between non-hydrogen atoms of different ligands was calculated using the pair fit command. […]

Pipeline specifications

Software tools PDB2PQR, NAMD, VMD, Colvars, XDS, CCP4, Coot, PRODRG, PHENIX, PyMOL
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma
Chemicals Aminoglycosides