SUMMARY (Project 2) A key theme of this Program Project Grant is that ATP-binding cassette transporter A1 (ABCA1) makes specific contacts with both lipid-free apolipoprotein A-I (APOA1) and certain high-density lipoprotein (HDL) subspecies to export lipids from cells to generate mature HDL. This removal of cholesterol from the artery wall protects against cardiovascular disease. Project 2 picks up on Project 1’s observation that small HDLs are a subspecies that promotes cholesterol efflux via ABCA1 and that it is especially important for cardioprotection in diabetes. In addition, we found that the presence of HDL’s second-most abundant protein, apolipoprotein A-II (APOA2), also promotes ABCA1-mediated cholesterol efflux to fully lipidated HDL particles. These observations counter the commonly accepted idea that only lipid-poor apolipoproteins can engage ABCA1. Taking a mechanistic and functional approach, the main goal of Project 2 will be to understand how lipidated forms of HDL can interact with ABCA1 to promote lipid efflux. The central hypothesis is that the APOA1 in small HDLs, and in those containing APOA2, undergoes a conformational change, possibly in its C-terminus, that allows it to interact with ABCA1 (called the “Flipped Ends’ hypothesis). Aim 1 will use high-resolution cryo- EM and other structural techniques to understand how APOA2 affects APOA1’s structure and its functionality in cholesterol efflux assays. We will work hand-in-hand with Project 3 to not only model APOA1/A2 interactions but also to visualize how those particles interact with ABCA1. The specific APOA2 sequences involved will be identified with an eye toward developing therapeutics that stimulate the transfer of cholesterol from ABCA1 to lipid-containing HDL particles. Aim 2 will use novel mutations of APOA1 that alter the sizes of HDL particles along with sophisticated particle reconstitution and structural techniques, such as hydrogen-deuterium exchange, to identify structural features in small HDL that allow it to engage with ABCA1. We will also identify and structurally characterize HDL subparticles in human plasma that can interact with ABCA1. Therapeutics that boost levels of lipid-poor APOA1 as substrates for cholesterol efflux by ABCA1 are being evaluated in clinical trials. Our work suggests that it may be possible to design therapies that not only increase cholesterol efflux by ABCA1 to lipid-poor acceptors but also to preformed HDL particles—a pool with much higher capacity as it comprises the lion’s share of circulating APOA1.