Project Summary Peroxisomal ABC transporters like ABCD1, ABCD2, and ABCD3 shuttle very long chain fatty acids (VLCFAs), branched chain fatty acids (BCFAs), and bile acid precursors into peroxisomes. Their functional impairment leads to severe neurological and metabolic pathologies stemming from disrupted phospholipid and fatty acid metabolism, including X-linked Adrenoleukodystrophy (X-ALD), both the most common leukodystrophy and most common peroxisomal disorder that is caused by mutations in ABCD1 and for which no cure exists, and bile acid synthesis defects and liver disease stemming from impaired ABCD3 function. While additional roles for ABCD transporters in a wider array of disease pathways continue to be uncovered, the underlying mechanisms governing their substrate recognition, transport, and transport regulation remain poorly understood. The long- term objectives of this project are to gain insight into peroxisomal ABCD transporter function and regulation in molecular detail. We will use a combination of biochemical and cell biological tools, high resolution structural analysis by cryo-electron microscopy, and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy to reveal the functionally relevant structural features and conformational states used by ABCD transporters in fatty acid translocation, how they may be altered by ABCD1 mutation in X-ALD, and how ABCD1 is mechanistically distinct from ABCD2 and ABCD3 despite their functional overlap. Specific Aim 1 deals with the development and utilization of in vitro assays for determining substrate specificity profiles and transport properties of ABCD1, ABCD2, and ABCD3. Specific Aim 2 deals with obtaining high resolution structural information of ABCD1, ABCD2, and ABCD3 in functionally relevant states in a physiological lipid environment through cryo-EM analysis. Specific Aim 3 deals obtaining information on the structural dynamics of ABCD1 through CW-EPR studies. Our results will provide fundamental insights into peroxisomal ABC transporter functioning that can be exploited for ABCD1 targeted diagnostic and therapeutic tools to improve X-ALD patient outcomes, provide a framework for the design and development of chemical probes to study ABCD family function, and generate reliable in vitro and in silico tools to accelerate drug development/discovery efforts targeting them.