Characterizing Structural and Dynamical Impacts of Agonist- and Antsgonist-Stabilized Mutants of the Estrogen Receptor

Abstract

The estrogen receptor (ER) is a ligand-regulated transcription factor belonging to the nuclear receptor superfamily and is involved in numerous physiological and disease states, most notably breast cancer. Extensive mutational studies have identified several point mutations within the binding domain that stabilize agonist and antagonist functional states and allow for structural characterization of the ER in these states. Characterizing differential dynamics of these mutants and wildtype ER, therefore, can be used as a way to understand the nature of conformational changes induced by binding of each class of ligands and to characterize the impact of specific residues on the overall functional state of the receptor.Equilibrium molecular dynamics (MD) simulations of the agonist-stabilized Y537S structure have revealed the formation of a new hydrogen bond (S537-D351), but challenged the role of solvent exposure for hydrophobic residues in the enhanced stability of the mutant structures, as has been suggested based on x-ray structures. Furthermore, we have observed the formation of transient hydrogen bond networks surrounding the base of critical helix 12 (H12) that were not observable from static structures. Steered MD (SMD) simulations have also shown different unbinding modes of H12 upon applying a force perpendicular to the length of the helix.Analysis of the antagonist-stabilized L536S structure indicated that the mutation resulted in a 157-degree rotation of the mutated residue, repositioning the preceding flexible loop into an ordered conformation against the protein surface. Attempts to replicate this effect in silico by mutating the WT structure of ER-bound hydroxytamoxifen and simulating for 20 ns resulted in only a partial rotation of the loop. Applying a harmonic force to slowly complete the rotation of the S536 psi angle revealed a significant energy barrier to obtain the final mutant conformation.