UCSF

Proteins are the macromolecular machines responsible for many of life’s fundamental processes. As extended polymers of amino acids, proteins fold into unique three-dimensional shapes in order to carry out their biological roles. Understanding the connection between protein structure and function has been an active area of research for much of the last century, and throughout that time the primary method of structure determination has been X-ray crystallography. Recent advances in the field of cryo-electron microscopy (cryoEM), particularly with respect to electron detectors and algorithms for image processing, have allowed the technique to reach resolutions that rival X-ray diffraction studies. Without the requirement of a crystallized sample, cryoEM opens the possibility of visualizing previously intractable protein targets as well as flexibility and motion within those proteins. My thesis focuses on the application of single particle cryoEM to study membrane proteins in lipidic environments and the conformational changes they undergo.

In the first chapter, I present a series of structures of the Bos taurus multidrug resistance protein 4 (MRP4). MRP4 is an ATP binding cassette transporter belonging to the ABCC family and is responsible for the rapid efflux of multiple endogenous and exogenous substrates. Included among these substrates are prostaglandins, a group of biologically active lipid molecules tied to physiological processes as diverse as inflammation and vasoactivity, and whose dysregulation is implicated in pathologies such as cancer and thrombosis. While MRP4 plays a crucial role in localized cell-to-cell signaling as the sole prostaglandin exporter identified in eukaryotes, the molecular details of it’s transport activity are poorly understood. Using single particle cryoEM, I determined five high resolution structures of MRP4 along various steps of the substrate transport cycle. These structures reveal for the first time the basis of MRP4’s affinity for prostaglandins and other organic anions, how substrate binding can stimulate MRP4’s basal ATPase activity several fold, and the conformational changes required for substrate transport. These results broaden our understanding not only of MRP4, but also closely related members of the ABCC family.

In the second chapter, I and others describe the novel structure of Arabidopsis thaliana AKT1, a hyperpolarization-activated voltage-gated K+ channel responsible for K+ uptake by the plant’s roots. AKT1 is a Shaker-like channel, and forms homo-tetramers that are under multiple, redundant levels of regulation. Our single particle cryoEM analysis revealed a potential novel form of autoinhibition via a disulfide bond between a soluble N-terminal helix and C-linker. The orientation of this covalent linkage sterically hinders the activation of AKT1’s voltage-sending domain and induces a dramatic restructuring of the tetramer from a C4-symmetric channel into a C2-symmetric one. The transitions between the two conformations of AKT1 found in the C2-symmetric channel resemble those of cyclic-nucleotide gated ion-channels upon cyclic nucleotide binding, suggesting a regulatory role for the previously unreported disulfide bond. Our proposed model of AKT1 autoinhibition provides insights into similar forms of regulation across other hyperpolarization-activated channels.