On early endosomes (EE), the Early Endosomal Antigen 1 (EEA1) functions as a tether for incoming vesicles through binding to the small GTPase Rab5. However, little is known about the mechanism of interaction of EEA1 with the EE membrane and the organization and mechanical properties of the bound protein. In this work, we investigated the binding of the full-length coiled-coiled EEA1 to EE by using the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) combined with a supported lipid bilayer (SLB). We reconstituted EEA1 binding to lipid membranes corresponding to the EE composition, including its lipid partner, phosphatidylinositol 3-phosphate (PI(3)P). Furthermore, we studied the effect of different lipids present on EE membranes and tested the binding specificity and kinetics in real time with the QCM-D. One of the main findings is that, although PI(3)P is the key lipid for the EEA1-membrane interaction, cholesterol and phosphatidylserine greatly influenced the kinetics and extent of EEA1 binding to membranes. Moreover, we employed the available models to analyze the QCM-D data with emphasis on the viscoelastic model. Our results show that, in our in-vitro system, EEA1 is not fully extended and/or highly packed, but mainly in a bent, distorted conformation. This study complements previous works employing in vitro assays and the QCM-D demonstrating as well the ability to reconstitute complex biomimetic membranes containing inositol phospholipids for the study of EE-related events such as the EEA1 binding.
