Membrane Bound Structures Of Peripheral Membrane Binding Proteins Tim3 And Tim1 Produced By Molecular Dynamics Informed Analysis Of X-Ray Scattering Experiments

Lipid binding and associating proteins are necessary components of cell signaling pathways historically overlooked for more amenably characterized protein-protein interactions. As peripheral membrane binding proteins attract more attention, reliable structural methods are needed to elucidate the protein-lipid interactions that facilitate their function. Traditional methods such as crystallography or NMR have produced structures of many peripheral membrane binding proteins in isolation, bound to a single lipid, or in a lipid cubic phase but not in complex with full lipid membranes. X-ray reflectivity provides structural characterization of lipid monolayer associated proteins assuming a known structure of the desired protein has already been obtained. Depending on the experimental conditions of the given structure, it is possible this structure is representative of the membrane associated structure. In our studies of three members of the T-cell Immunoglobulin Mucin (TIM) family of proteins, involved in the recognition of the apoptotic cellular signal phosphatidylserine (PtdSer) in lipid membranes, the crystal structure was only representative for TIM4 and not TIM1 or TIM3. TIM1 was crystallized without PtdSer in a closed conformation that cannot represent the PtdSer bound state and TIM3 has much lower affinity resulting in a weak x-ray reflectivity signal. We developed data analysis methods employing molecular dynamics to refine the structures of TIM1 and TIM3 to better represent their membrane bound conformations. The newly obtained structures provide much improved fits of the data and highlight protein-lipid interactions that can explain the differences in binding affinity between the TIM protein family members.