Molecular Dynamics Simulation
Molecular dynamics (MD) simulations were performed and analyzed with the Molecular Operating Environment (MOE) (Chemical Computing Group, Canada) and NAMD 2.7 suite [http://www.ks.uiuc.edu/Research/namd] (Phillips et al., 2005). For molecular dynamics simulation with the antibody-ligand complexes, starting coordinates were from homology modeled structures. Hydrogen atoms were added to the starting structures using the LigX protonation tool of the MOE 2016.08 suite. Using the solvation algorithm in MOE, a 10 Å region around the protein-ligand complex was immersed in a TIP3P water droplet such that protein atoms outside the droplet were fixed to dampen long range dynamic effect. Sodium cations were added for electroneutrality. Additional Sodium and Chloride ions were added to a final concentration of 0.15 M. This resulted in systems consisting of 18,780 atoms for the mAb1,17,788 atoms for mAb2, and 16,469 atoms for mAb3. Dynamics simulations were then performed using NAMD, with parameters from the Amber10:EHT forcefield set. Equilibration consisted of 15, 000 steps of conjugate gradient energy minimization, followed by 50 ps of MD with restraints applied to the protein to equilibrate the water. The systems were then warmed to 300 K over a total of 18 ns with a 50 K increment and equilibration at each temperature step. This was followed by unrestrained production run of 30 ns, which is enough for the determination of empirically relevant binding affinities (Salvalaglio, Zamolo, Busini, Moscatelli, & Cavallotti, 2009). The SHAKE algorithm was used, allowing a 2 fs time-step. Long-range electrostatics were treated via the particle mesh Ewald method. Trajectory analysis was carried out with the MOE analysis tool and in-house scripts.