Predicting Ligand Binding Affinity with Alchemical Free Energy Methods in a Polar Model Binding Site

Abstract

We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed relative binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 (phenol) and 1.1 (catechol) kcal/mol. To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0Å (diverse set), 1.8Å (phenol derived predictions) and 1.2Å (catechol derived predictions). We found that to predict accurate affinities and rank-orderings required nearnative starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe these results can help guide future improvements in physics-based absolute binding free energy methods.

Homologous ligands accommodated by discrete conformations of a buried cavity

“…Formed in the hydrophobic core of the protein, the resulting 150-Å 3 cavity is sequestered from solvent and is almost entirely apolar. Seminal studies by Matthews and colleagues demonstrated that this cavity can bind aryl hydrocarbons (20,21), and since then the cavity and related mutants have become model systems for ligand recognition (22)(23)(24)(25)(26)(27)(28)(29). Contributing to this status has been the commercial availability of thousands of likely ligands, many closely related to one another.…”

Separated topologies—A method for relative binding free energy calculations using orientational restraints

“…These orientational restraints are commonly applied in absolute binding free energy calculations. 22,23,12,24 In essence, two absolute binding calculations are performed at once, in different directions, and overlapping in the middle. The path (representing transformation G site in Figure 1) is shown in Figure 2.…”

“…Even very similar ligands in simple binding sites often adopt different orientations and the kinetic barriers between orientations can be significant enough that ligands will not reorient during relative free energy calculations using the standard approaches reviewed earlier (cf. the difference between relative transformations with phenol and catechol in Boyce et al (2009) 12 ). Also, even if ligands eventually reorient, this step can be rate limiting to converging the relative binding free energy, meaning that using a cycle which does not require reorientation will be more efficient.…”

“…In the single topology approach, there is one ligand molecule in all simulations, which morphs from being in state A (having a particular chemical structure and interaction parameters), to a different state B, where the structure and interaction parameters are different. [4][5][6][7][8][9][10][11][12][13] In some transformations, there could be atoms on A that have no direct analog on B. During the computational morphing process, these atoms will have their non-bonded interactions decoupled from the rest of the sysa) Author to whom correspondence should be addressed.…”

“…12 Similarly, there may be atoms that are unique to B. When transforming A into B, these atoms would begin as dummy atoms and become coupled to the system (protein plus solvent) during the computational morphing process.…”