(380f) The Role of Intrinsically Disordered Proteins in Membrane Curvature Sensing

Protein-lipid interactions in biological membranes, such as membrane curvature sensing, are essential for a variety of cellular processes. In clathrin-mediated endocytosis (CME), membrane curvature sensing is necessary for the efficient assembly of proteins at highly curved membrane sites. Established mechanisms of curvature sensing rely on proteins with specific structural features such as amphipathic helices. In contrast, we have recently discovered that intrinsically disordered proteins (IDPs), which lack a defined three-dimensional fold, can also be potent sensors of membrane curvature. This ability of IDPs to sense curvature arises from two key physical features – a high degree of conformational entropy and a high net negative charge. Binding of such IDPs to membrane surfaces results simultaneously in a decrease in conformational entropy and an increase in electrostatic repulsion by anionic lipids. Here we show that each of these effects gives rise to a distinct mechanism of curvature sensing. Specifically, as the curvature of the membrane increases, the steric constraint that it imposes on the conformation of the IDP is reduced, leading to an entropic preference for curved membranes. At the same time, increasing membrane curvature increases the average separation between anionic amino acids and anionic lipids, leading to an electrostatic preference for curved membranes.

To examine curvature sensitivity by IDPs, we engineered various truncation and chimeric mutants that were derived from the endocytic adaptor proteins AP180, Epsin1, and Amphiphysin1. Using Monte Carlo simulation and quantitative in vitro fluorescence techniques, our results demonstrate that long IDP chains with relatively low net charge sense membrane curvature predominately through the entropic mechanism, while shorter, more highly charged IDP chains rely largely on the electrostatic mechanism. We also demonstrate that IDPs can sense membrane curvature in live cells and that full-length endocytic proteins, which contain both structured curvature sensors and disordered regions, are more than twice as curvature sensitive as their respective structured domains alone.

Interestingly, Amphiphysin1 and Epsin1 are also able to impart substantial curvature sensitivity upon clathrin itself. Specifically, in the presence of these adaptor proteins, clathrin becomes a potent sensor of curvature, with even greater curvature sensitivity than that of adaptor proteins themselves, suggesting a synergistic interaction between the clathrin and its adaptor proteins. Similarly, when an artificial membrane-binding tag was used to assemble clathrin directly on membrane surfaces, clathrin’s curvature sensitivity decreased substantially, suggesting that the interaction with adaptor proteins strongly promotes curvature sensing by clathrin. These results indicate a cooperative binding effect, where the avidity between Amphiphysin1 and clathrin increases as the density of Amphiphysin1 on the membrane surface increases. These findings elucidate the synergy between structured and disordered protein domains in membrane remodeling and curvature sensing during CME.