Mechanism of recognition of phosphatidylserine by peripheral immunological proteins

Current: Shou-Ting Hsieh
Alumni: Zhiliang Gong, Daniel Kerr, Tiffany Suwatthee

Phosphatidylserine (PS) is the most abundant negatively charged lipid in cell membranes, constituting 10-20% of cell lipids. Normally it resides in the inner leaflet of the cell membrane. Exposure of PS to the outer leaflet is an important signal that plays vital roles in numerous cellular functions and disease states, including blood coagulation, cell fusion, apoptosis, autoimmune responses, chronic viral infection, etc. Correspondingly an array of more than 30 PS recognizing proteins has been identified. How this seemingly generic signaling of PS exposure could be involved in such diverse physiological processes is unclear. We take particular interest in how PS interacts with a group of peripheral immunological proteins involved distinct functions in the innate and adapted immune systems. Specifically we have focused on four proteins, T cell/transmembrane immunoglobulin mucin protein 1, 3, 4, and MFG-E8. Using a combination of fluorescence spectroscopy, x-ray surface scattering, x-ray fluorescence, and molecular dynamics, we seek to understand how exactly these proteins recognize PS differently, and how such difference could lead to distinct functional outcomes.

A tryptophan fluorescence assay was developed to measure the binding of TIM proteins to lipid membranes. All of the TIMs have a Tryptophan at the surface which burrows into the membrane when binding occurs. Tryptophan is fluorescent and its emission changes when it’s in water or in a hydrophobic environment like the tails of lipids in a membrane.

Interfacial x-ray scattering can determine the membrane bound orientation of each of the Tim proteins to identify the molecular mechanisms that drive their unique recognition properties.

MD simulations generate protein structures used to fit x-ray scattering data and determine the orientation of the proteins with the membrane. TIM1 and TIM3 required MD to get correct orientations. TIM1’s crystal structure is not in a bound conformation while TIM3’s NMR structure lacks a number of residues. MD allowed us to resolve these hurdles.

Recently we’ve been developing tools to measure Calcium fluorescence on membranes using x-ray fluorescence. Early results indicate the TIM proteins bring calcium to the membrane interface when bound. X-rays are transmitted at various angles to construct a profile of how calcium is distributed at the surface of the lipid membrane. Higher fluorescent yield means there’s more calcium, upon addition of TIMs we see the yield increase. We believe this accumulation of calcium is important in the chemistry of the TIMs interaction with lipid membranes though right now we don’t fully understand the mechanism behind it. Using this technique with the brilliant x-ray at the Advanced Photon Source we’ve been able to qunatify calcium accumulation on the surface.