Computing Absolute Binding Affinities by Streamlined Alchemical Free Energy Perturbation (SAFEP)

Abstract

Free Energy Perturbation (FEP) is a powerful but challenging computational technique for estimating differences in free energy between two or more states. This document is intended both as a tutorial and as an adaptable protocol for computing free energies of binding using free energy perturbations in NAMD. We present the Streamlined Alchemical Free Energy Perturbation (SAFEP) framework. SAFEP shifts the computational frame of reference from the ligand to the binding site itself. This both simplifies the thermodynamic cycle and makes the approach more broadly applicable to superficial sites and other less common geometries. As a practical example, we give instructions for calculating the absolute binding free energy of phenol to lysozyme. We assume familiarity with standard procedures for setting up, running, and analyzing molecular dynamics simulations using NAMD and VMD. While simulation times will vary, the human tasks should take no more than 3 to 4 hours for a reader without previous training in free energy calculations or experience with the Colvars Dashboard. Sample data are provided for all key calculations both for comparison and readers' convenience.

Comparison of Receptor–Ligand Restraint Schemes for Alchemical Absolute Binding Free Energy Calculations

“…By moving away from the six anchor points of the VBA, this was intended to simplify the selection of stable and efficient restraints. The “distance to bound configuration” (DBC) restraint is also intended to simplify restraint selection and to minimize the variance of free energy estimates for removing the ligand’s intermolecular interactions. − This is achieved by directly restraining the RMSD of a subset of the ligand coordinates within the frame of reference of the binding site in order to optimally restrict the accessible configurational volume as the ligand intermolecular interactions are removed. However, because this scheme couples the internal and external degrees of freedom of the protein and ligand, there is no simple way to calculate the free energy of releasing the noninteracting ligand to the standard state.…”

Propofol directly binds and inhibits skeletal muscle ryanodine receptor 1 (RyR1)

“…Free energy perturbation (FEP) molecular dynamics simulations were conducted according to the Streamlined Alchemical Free Energy Perturbation (SAFEP) methodology, described in detail in (21). We followed the set-up and analysis procedure described in (20). Briefly, SAFEP uses a limited set of restraints on the ligand to maintain its bound conformation during alchemical transformations and improve sampling of states that most contribute to the binding free energy.…”

“…From the dose response curve for ryanodine we found that 1 µM ryanodine resulted in the maximum Fura-2 340:380 nm ratio, and as a result this concentration was used to determine the inhibition, if any, caused by different concentrations of propofol. The various propofol concentrations (2,5,10,20,50,100,200, 300 µM) was added to 1µM ryanodine. The 340/380 nm ratios were measured using the same method in Experiment 1.…”

“…Next, we identify several propofol binding sites through analogue photoaffinity labeling (17)(18)(19). Finally, we computationally predict the binding affinity of propofol to one of the identified binding pockets using streamlined alchemical free energy perturbation molecular dynamics (SAFEP MD) (20,21), suggesting that propofol may be bound to this site at clinically tractable concentrations. Taken together, our results suggest that propofol binds and inhibits RyR1 opening at clinical concentrations and may thereby prevent the clinical manifestations of MH even with exposure to triggering agents such as the volatile anesthetics.…”