Intermediate states in the binding process of folic acid to folate receptor α: insights by molecular dynamics and metadynamics

Folate receptor alpha (FR alpha) is a cell surface, glycophosphatidylinositol-anchored protein which has focussed attention as a therapeutic target and as a marker for the diagnosis of cancer. It has a high affinity for the dietary supplemented folic acid (FOL), carrying out endocytic transport across the cell membrane and delivering the folate at the acidic pH of the endosome. Starting from the recently reported X-ray structure at pH 7, 100 ns classical molecular dynamics simulations have been carried out on the FR alpha-FOL complex; moreover, the ligand dissociation process has been studied by metadynamics, a recently reported method for the analysis of free-energy surfaces (FES), providing clues on the intermediate states and their energy terms. Multiple dissociation runs were considered to enhance the configurational sampling; a final clustering of conformations within the averaged FES provides the representative structures of several intermediate states, within an overall barrier for ligand escape of about 75 kJ/mol. Escaping of FOL to solvent occurs while only minor changes affect the FR alpha conformation of the binding pocket. During dissociation, the FOL molecule translates and rotates around a turning point located in proximity of the receptor surface. FOL at this transition state assumes an "L" shaped conformation, with the pteridin ring oriented to optimize stacking within W102 and W140 residues, and the negatively charged glutamate tail, outside the receptor, interacting with the positively charged R103 and R106 residues, that contrary to the bound state, are solvent exposed. We show that metadynamics method can provide useful insights at the atomistic level on the effects of point-mutations affecting functionality, thus being a very promising tool for any study related to folate-targeted drug delivery or cancer therapies involving folate uptake.