Decoding the structure-dependent binding of phthalate esters to GPER: A combined spectroscopic and multiscale computational study

An integrated approach combining multispectral techniques and multiscale simulations systematically reveals PAE–GPER interactions from molecular details to conformational changes. • The results show that substituent chain length and spatial distribution jointly regulate binding modes and significantly influence complex stability and protein conformational dynamics. • The established structure–interaction relationships provide a theoretical basis for endocrine risk assessment of PAEs and the rational design of safer alternatives. The G protein-coupled estrogen receptor (GPER) orchestrates numerous physiological processes, yet the structural determinants governing its interaction with ubiquitous endocrine-disrupting phthalate esters (PAEs) remain poorly defined. Here, we integrated multispectral characterization with computational modeling to elucidate the PAE–GPER interaction landscape. Virtual screening of 23 PAE congeners established that binding affinity was critically modulated by alkyl chain length, with molecular docking implicating Glu-275 and Gln-138 as essential anchoring residues. Molecular dynamics simulations revealed the formation of stable complexes, evidenced by a reduced radius of gyration (Rg) indicative of protein compaction, while solvent-accessible surface area (SASA) and root-mean-square fluctuation (RMSF) analyses demonstrated compound-specific remodeling of the protein microenvironment—findings corroborated by three-dimensional fluorescence spectroscopy. Thermodynamic profiling, MM-PBSA free energy calculations, and reduced density gradient (RDG) analyses converged to show that binding was spontaneous and predominantly mediated by van der Waals interactions and hydrophobic effects, with hydrogen bonding constituting a central stabilizing interaction. Steady-state fluorescence confirmed a static quenching mechanism, and Förster resonance energy transfer (FRET) analysis yielded donor–acceptor distances of 2.65–3.31 nm, situating these interactions within the optimal energy-transfer regime (0.5 R₀ –1.5 R₀ ). Molecular electrostatic potential (MEP) mapping further highlighted synergistic hydrophobic and electrostatic contributions to recognition. Finally, Fourier-transform infrared (FT-IR) spectroscopy detected ligand-induced secondary-structure rearrangements, exemplified by a 3.03% increase in β-sheet content upon dibutyl phthalate (DBP) binding. Collectively, these data identify steric constraints and hydrophobic complementarity as pivotal determinants of PAE–GPER engagement, providing a mechanistic framework for evaluating the endocrine-disrupting potential of structurally diverse environmental contaminants. Download: Download high-res image (325KB) Download: Download full-size image