Computational protocol: Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

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Protocol publication

[…] All molecular dynamics (MD) simulations were performed using the GROMACS (Groningen Machine for Chemical Simulation) package, version 3.3.3 , using the GROMOS 54A7 force field for peptides. The simple point charge (SPC) water model was used to describe the solvent water. All simulations were performed under periodic boundary conditions in a rectangular box. The dimensions of the box were chosen such that the minimum distance to the box wall was at least 1.0 nm. A twin-range method was used to evaluate the non-bonded interactions. Interactions within the short-range cutoff of 0.9 nm were updated every step. Interactions within the long-range cutoff of 1.4 nm were updated every 3 steps together with the pair list. A reaction field correction was applied using a relative dielectric constant of εr  =  78.5 to minimize the effect of truncating the electrostatic interactions beyond the 1.4 nm long range cutoff . The SHAKE algorithm was used to constrain the lengths of the covalent bonds. The geometry of the water molecules was constrained using the SETTLE algorithm . In order to extend the timescale that could be simulated, explicit hydrogen atoms in the protein were replaced with dummy atoms, the positions of which were calculated each step based on the positions of the heavy atoms to which they were attached. This eliminates high frequency degrees of freedom associated with the bond angle vibrations involving hydrogens, allowing a time step of 4 fs to be used to integrate the equations of motion without affecting thermodynamic properties of the system significantly . The simulations were carried out in the NPT-ensemble at T  =  300 K, and P  =  1 bar. The temperature and pressure were maintained close to the reference values by weakly coupling the system to an external temperature and pressure bath using a relaxation time constant of 0.1 ps and 0.5 ps, respectively. The pressure coupling was semi-isotropic. Data was collected every 40 ps for analysis. Images were produced using VMD .The protonation states of all ionizable residues were assigned as described previously . Specifically, at neutral pH three histidine residues, His 149 and the catalytic histidines His583 and His1228, were doubly protonated. Residues 1 to 32, 1273 to 1284, and 58 residues of the subunit linker were not observed crystallographically. The termini were not modeled, as there is no information on the structure of these regions. These regions also contained the signal anchor and the His-tag sequences. The subunit linker was also omitted from the simulations. This was because it has been shown experimentally that P-gp is fully functional in the absence of the linker –. In addition, the ability to model loops accurately is poor for loops containing more than 8 to 12 amino acids , . Including the linker would, therefore, risk biasing the simulations unnecessarily. Nevertheless, the termini of the peptide chains were neutralized by artificially protonating the C-termini and deprotonating the N-termini to avoid the introduction of inappropriate charges within the protein, as described previously by O’Mara and Mark .The P-gp crystal structure (PDBid 3G5U-a) was inserted into a lipid bilayer containing a 10:1 ratio of POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine) and cholesterol . One molecule of ATP was placed in close proximity to the Walker A motifs of each NBD, in the orientation given by the MalK crystal structure . Mg2+ was placed in the vicinity of each of the four Mg2+ binding sites in the NBDs determined from previous MD simulations . The system was solvated with SPC water and sufficient Cl− counterions added to ensure the overall charge neutrality of the system. Further ions were added to adjust the concentration of the solution to 150 mM NaCl and 1.5 mM MgCl2.The system was equilibrated as follows. Initially 500 steps of steepest descent energy minimization were performed to relax the solvent with all the heavy atoms within P-gp restrained using a harmonic potential with a force constant of 1000 kJ·mol−1·nm−2. Then a series of 1 ns simulations were performed in which the force constant was progressively reduced (1000, 100, 50 kJ·mol−1·nm−2). To allow ATP to fully associate with the Walker A and B motifs, each system was simulated for a further 7 ns in which the backbone atoms of P-gp were restrained using a harmonic potential with a force constant of 50 kJ·mol−1·nm−2. The restraints were then removed and new velocities were assigned. Initially, four independent MD simulations (Runs A to D) were performed each for a period of 90 ns.In the first 30 ns of Runs C and D1, the ATP molecule bound to the NBD2 Walker A motif and to the NBD1 Signature motif, inducing closure of the NBD2 ATP binding site. This conformation was maintained throughout the remainder of the simulation (90 ns). To examine this state in more detail, Run D1 was branched after 30 ns into three separate simulations. New velocities were assigned and each system was simulated for a further 90 ns (Runs D2 to D4). The original Runs C and D1 were also extended for an additional 30 ns so each system was simulated for a total of 120 ns. gives a summary of all simulations performed, together with the sequence motifs binding ATP at each of the ATP binding site at the end of each simulation. […]

Pipeline specifications

Software tools GROMACS, VMD
Application Protein structure analysis
Chemicals Adenosine Triphosphate