Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)
Protein-lipid interactions constitute a very important class of biological interactions critical for multiple cell and tissue functions. It is believed that most lipid-protein interactions are very weak, with affinities in the 1mM-1M range. Here we study the interactions of multiple protein lipid binding domains with lipid membranes containing signaling lipids known as phosphatidylinositol phosphates (PIPs) using a new mechanically transduced immunosorbent assay (METRIS). We demonstrate that this assay can measure extremely weak interactions at PIP bilayer concentrations below 1%, which is close to the biological lipid concentration regime. In particular, we have studied the interaction of DrrAWT, DrrAK568A, PH-{delta}, and 2XFYVE as well as previously unexplored lipid binding domains such as Auxilin 1 (PTEN) and Auxilin 2 (GAK) against a wide palette of PIPs. Our results confirm that each of these domains interacts specifically with a PIP partner. In the case of Auxilin 1 and Auxilin 2, both proteins in the Clathrin endocytotic pathway, we find that their PTEN-like domain interacts specifically yet with ultra low affinity with PI3P and PI4P respectively. We have also found a new unknown medium-high affinity interaction between GAK with PI34P2. Our work, thus, provides a direct and robust method to measure and catalog protein lipid interactions which are important in many processes such as signaling and membrane sculpting. Furthermore, this assay can be extended in a straightforward manner to study other interactions such as ligand-receptor or antibody-antigen.