Multiple dynamically-coupled binding sites on human serum albumin regulate estradiol's nonlinear binding

Circulating estradiol is predominantly protein-bound, with human serum albumin (HSA) serving as its major carrier. While traditionally considered a carrier with low affinity and readily reversible binding at a single site, the molecular details and kinetics of estradiol-HSA interactions remain incompletely understood.

We employed equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy to characterize estradiol-HSA interactions. Surface plasmon resonance (SPR) was used to elucidate the kinetics of estradiol's association and dissociation with HSA. Structural and energetic features of binding were investigated using molecular docking and structure network analyses.

Binding isotherms generated using equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy revealed nonlinear asymmetric binding with apparent Kd that varied as a function of estradiol and HSA concentrations, inconsistent with canonical model of low-affinity, single-site interaction characterized by a fixed Kd. Kinetic analyses by SPR revealed initial rapid association dynamics followed by a slower second phase. Molecular modeling identified a high-affinity estradiol-binding pocket in Sudlow's Site I and two additional low-affinity sites within a highly interconnected hub of structural blocks. Spatially coordinated conformational rearrangements accompanying estradiol partitioning into the high-affinity pocket of Sudlow's Site I and two additional moderate-affinity sites suggest an allosterically-coupled binding architecture that enables albumin to actively regulate estradiol bioavailability across a broad, physiologically relevant concentration range.

Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.