Structural and Functional Methods To Understand ABC Transporters Conformational Change and Regulation

Abstract

ABC (ATP-binding cassette) transporters are integral membrane proteins that play a vital role in cellular transport processes by shuttling a wide range of substrates across cellular membranes. These transporters undergo complex conformational changes during their functional cycle, which are tightly regulated to ensure precise substrate transport. Understanding the structural dynamics and regulatory mechanisms of ABC transporters is of paramount importance, as they are implicated in various physiological processes and are targets for drug development. The major focus of this dissertation is to present structural and functional methods employed to elucidate the intricate conformational changes and regulatory mechanisms governing ABC transporters. We revealed an atomic resolution cryo-electron microscopy model, that has provided crucial insights into the dynamic nature of the ABC-C transporter family. This method has enabled the visualization of the Yeast Cadmium Factor (YCF1) transporter, at a different phosphorylation stage important for its regulation. We successfully identified a crucial phosphorylation site, as well as relevant amino acids and domain interactions. Furthermore, we delve into the biochemical and biophysical techniques to probe the functional aspects of the ABC transporters, not only with YCF1 but also sampling the ABC-B family. Substrate binding assays, ATPase activity, thermostability and cellular assays, aided our group to expand on the kinetics and energetics of substrate transport. This work describes a structural approach, such as single-molecule imaging and computational modeling, and examines sophisticated biochemical regulation of ABC transporters. The approaches implemented here could potentially be used for the development of novel therapeutic targets for ABC transporters related diseases as well as expand the full turnover cycle of this important class of proteins.