Structural basis of apoptotic scrambling - ABSTRACT Every day ~50 billion cells undergo apoptosis, or programmed cell death. Efficient recognition and clearance of these cells is critical for tissue homeostasis in physiology and for its recovery following disease. Initiation of the apoptotic cascade triggers activation of phospholipid scramblases that externalize the lipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane. Recognition of PS by dedicated receptors (PSRs) on immune cells is the first and critical step in the clearance of apoptotic cells. Immune cells with activated PSRs create an immunosuppressive environment that can be exploited by pathogens exposing PS in an immune-camouflage strategy called apoptotic mimicry. Recent studies implicated members of the XKR protein family in apoptotic scrambling. Dysfunction of XKR proteins results in an inflammatory environment and autoimmune disorders while their uncontrolled activation favors oncogenesis and facilitate viral entry. Thus, it is critical to elucidate the molecular mechanisms of XKR function and regulation to understand their physiological roles and their association with pathology. Currently, only structures of non-functional XKR proteins are available, hindering our understanding of how these protein mediate PS externalization. In preliminary experiments we show that two XKR homologues, CED-8 from C. elegans and human XKR4, scramble lipids. Using cryogenic electron microscopy, we determined the 3.55 Å resolution structure of hXKR4 in a novel, likely active conformation, providing insights into a potential mechanism for lipid transport. In our 1st aim, we propose to determine the structures of hXKR4 and CED-8 in different conformations and functional states to determine the molecular bases of XKR activation. We will use our newly developed biochemical assays to probe and elucidate the functional implications of these structures. The physiological implications of these mechanisms will be tested in cell-based measurements. Our 2nd aim is to determine how XKR proteins scramble their surrounding membrane lipids. Using cryoEM we will directly visualize the hXKR4 and CED-8 scramblases in the context of a membrane to determine how they interact with and remodel the surrounding bilayer to enable lipid scrambling. Furthermore, we will investigate how changes in the physicochemical properties of the membrane support different functional states of these proteins. Our 3rd aim is to elucidate the regulatory mechanisms of XKR activity. Using in vitro and cell-based functional assays, we will determine whether and how hXKR4 and CED-8 are directly regulated by cellular factors, such as processing by apoptotic caspases, phosphorylation, or interactions with of a peptide derived from the nuclear DNA repair protein XRCC4. Collectively, our studies will lay the foundation for future investigations of the molecular underpinnings of the XKR scramblases in vivo.
