Title: Using Solid-state NMR to Study Protein Binding to Phosphatidylinositol Phosphates in Lipid Bilayers
Abstract: Phosphatidylinositol phosphates (PIPs) are eukaryotic membrane lipids that tightly regulate cellular processes such as cellular growth, metabolism, immunity, and development, through direct interactions with partner proteins. In this work, we seek to directly observe the structure and dynamics of PIP3 in lipid bilayers with and without kindlin-2 (K2), a partner protein activated by PIP3. We probe the effects of the anionic lipid phosphatidylcholine (PS), the divalent cation Ca2+, and cholesterol. We use solution and solid-state 1H, 31P, and 13C NMR combined with MD simulations to characterize the structure and dynamics of PIPs and the binding orientations of the K2 binding domains. Overall, we note the presence of PS restricts the lateral diffusion of PIPs. PIP3, like PS, forms dimers (and larger oligomers) in the presence of Ca2+ which explains differential changes in the rotational dynamics of the PIP headgroup phosphates. The K2 binding domains show numerous contacts with PIP3, many outside of the canonical binding site. The addition of cholesterol modifies the behavior of the lipids by themselves and greatly enhances the sensitivity of NMR experiments on the bound form of K2.Â