Structural and Functional Characterization of the Cytosolic Domains of the Copper Transporting P1B-ATPase CopB from Archaeoglobus fulgidus

Abstract

All cells attempt to maintain optimal levels of essential metal nutrients while eschewing toxic metal concentrations. To achieve this balance, metal homeostasis mechanisms have evolved to maintain appropriate levels (1). Of the many proteins which play a crucial role in metal homeostasis, the P-type ATPase family of transmembrane transporters is involved in the active transport of metal ions across the membrane using ATP hydrolysis as an energy source (2-5). P(1B) ATPases are found in all life kingdoms, from extremophilic archaea to mammals, and are a subgroup of P-type ATPases which are specific for heavy metal transport (4, 6). These transporters confer metal tolerance to archaea and bacteria and aid in metal efflux from the cytoplasm, thus conferring survival under conditions of high levels of metal in the environment. In humans defects in metal transport can result in severe hepatic and neurological disorders such as Menkes' and Wilson's disease, and thus understanding the molecular function of copper transporters is of paramount importance in human health (7). CopB, the 690 amino acid P(1B-3)-ATPase from Archaeoglobus fulgidus, is the central focus of this dissertation. CopB has a multiple cytosolic domains (ATP binding domain, actuator domain, and metal binding domain) in addition to 8 transmembrane helices (8).The transporter has a distinctive histidine rich N-terminal metal binding domain (MBD), which has been postulated to have a regulatory role in the transport process (9, 10). Although the MBD lacks sequence similarity to cytosolic metal binding domains belonging to homologous P-type ATPases, the preponderance of histidine residues suggests one or more metal coordination sites. Although membrane extracts of A. fulgidus CopB were shown to transport Cu(II), the metal binding affinity of the MBD, any potential metal dependent interactions with the ATP binding domain (ATPBD), and the structures of the cytosolic domains themselves remained unresolved when this research was initiated. Since then, the purpose of this study had been tripartite. First, I probed the relationship of the metal binding to the function of the N-terminal metal binding domain by investigating the metal binding properties of the MBD and the impact on its structure and inter domain interactions with the ATPBD and the A-domains, which has been seen in other homologous ATPases and shown to play an important regulatory role. The second focus was to determine the structure and characterize the ligand-induced conformational changes of the ATPBD. Finally I investigated the effects of some prominent Wilson's disease mutations which are conserved on the ATPBD of CopB, thereby using CopB as a model system. These studies have helped in understanding how heavy metal ATPases with distinct His-rich metal binding domains work in maintaining critical levels of metal ions across the cell membrane.