Ligand egress with Random Accelerated MD

In order to simulate the effects of E413G on the unbinding of glutamate from GluN2B, the system was modeled with Random Accelerated Molecular Dynamics (RAMD, originally known as Random Expulsion Molecular Dynamics) ⁴³. In the original implementation of this protocol, a random acceleration is applied to the ligand for a specified block of simulation time. If the ligand fails to move a minimum distance within that time, a new direction is randomly chosen. This technique was originally devised to explore long ligand access in tunnels with deeply buried active sites in cytochrome P450 in a tractable amount of simulation time ^43,44. By contrast, in the present study a new direction for the acceleration was chosen for each RAMD time-block, since the binding-site of glutamate in GluN2B is located very near the surface. This strategy was chosen to enable a more thorough exploration of ligand egress from the binding-site. The RAMD simulations ended when the ligand was 10 Å from the protein center-of-mass (COM).

A number of parameters were optimized in order to use RAMD as a reliable tool for distinguishing the effect of the mutant vs the wild-type protein. These included the magnitude of ligand acceleration, enforced randomization of the acceleration vector, and starting co-ordinates of the protein/ligand complex. The results of this optimization are discussed in the Supporting Information.

The following protocol was chosen for RAMD on glutamate; 30 different simulations were initiated from a random snapshot of non-accelerated MD (10-20 ns) of the protein/ligand complex. Different initial coordinates were used to reduce any bias that could be introduced by a starting conformation more propitious for ligand egress. A constant acceleration of 0.225 kcal.Å.amu.mol⁻¹ was applied to all atoms of the ligand, with a new direction randomly chosen every 0.2 ps (100 steps). Each simulation was run until the ligand was at least 10 Å from the protein center of mass. Such MD runs, took 1-10 ns and seldom exceeded 20 ns.

A similar RAMD protocol was also used to explore the egress of the cyclic glutamate derivative (2S, 4S)-2,4-piperidinedicarboxylate (PPC). Lower accelerations of 0.191 kcal.Å.amu.mol⁻¹ PPC, and a 15 Å COM cutoff were used to correct for the larger momentum and size of the ligand compared to glutamate. General CHARMM force field parameters for the ligands were assigned by analogy using the Paramchem website ^38,45–47.