BPS2026 – Structural origin of hTIM4 cooperative membrane binding for phosphatidylserine lipids using X-ray reflectivity combined with MD simulations

Human transmembrane immunoglobulin and mucin domain protein 4 (hTIM4) is a phosphatidylserine (PS)-binding protein expressed on the membrane surface of immune cells. The PS-binding pocket of hTIM4 is utilized to recognize PS lipids in the target cells, such as apoptotic cells. However, the level of PS exposure in cells can vary significantly depending on the cell status. Such variations suggest that level of PS exposure can potentially influence binding affinity of hTIM4. In this study, we aim to understand how membrane-binding of hTIM4 responds to the variation of PS exposure, and to identify the key molecular contacts of membrane binding. We first used a tryptophan fluorescence assay to examine how hTIM4 responds to the various PS exposure levels. The data show that hTIM4 exhibits cooperative binding to PS lipids in the membrane. In addition, the calcium ion concentration can modulate membrane-binding behavior at different PS mole fractions. To further elucidate the key molecular contacts for the observed cooperativity, we obtained a representative membrane-bound structure of hTIM4 by using X-ray reflectivity (XR) combined with molecular dynamics (MD) simulations. Our results identify potentially favorable electrostatic interactions between four positively charged residues in the vicinity of the binding pocket and negatively charged PS lipids. Among them, Lys44 and Arg49 are found to be in frequent contact with the PS lipids based on full membrane simulations. Our work reveals the structural origin of hTIM4 cooperative binding in spite of the presence of a single-binding pocket, demonstrating the capability of XR in combination with MD simulations in identifying the membrane-bound structure of the protein, which help in delineating the molecular details of the membrane-protein interactions.