Date of Award
Doctor of Philosophy
Dr. David Huffman
Dr. Blair Szymczyna
Dr. Gellert Mezei
Dr. Pamela Hoppe
Copper transporting ATPases (Cu-ATPases) are essential for mediating copper uptake, maintaining cellular copper levels and prevention of accumulation of toxic copper. The most studied copper ATPases are the Menkes and Wilson proteins in eukaryotes, CopA and CopB found in bacteria. The precise interaction of the metal binding domains with their copper donor, the association of each metal binding domain within the N-terminal portion of the protein, how they communicate with each other, and the eventual metal ion translocation across the membrane, is little known. The variation in number of the cytosolic metal binding domains may also offer clues on the structural and functional roles these Cu-ATPases play in organisms. In order to gain more insight on the structural organizations of copper ATPases, I have studied the N-terminal domains of three copper ATPases: (1) Wilson disease protein, (2) Dictyostelium discoideum Copper ATPases and (3), Plasmodium falciparum copper ATPase (PfCuP-ATPase).
The N-terminal portion of Wilson protein (ATP7B) is made of six metal binding domains which can receive copper(I) from a copper donor, HAH1, that forms a stable adduct with metal binding domain four. I engineered a chimera of this metal binding domain with the copper chaperone HAH1. When the chimera was denatured using guanidine hydrochloride, it unfolded in three phases. The first and second midpoint of unfolding is 1.37 M and 5.6 M GuHCl respectively. The midpoint of thermal unfolding is 78 ℃. The chimera was crystalized by hanging drop vapor diffusion at room temperature using Hampton crystallization screen.
Dictyostelium discoideum has three copper ATPases: ATP1, ATP7A and ATP3. The number and structural basis of the metal binding domains of these heavy metal transporters is little studied. Using bioinformatics tools, circular dichroism and solution NMR, I have identified two N-terminal domains of ATP1. Domain1 has MXCXXXC metal binding motif that is uncommon of PIB type Cu ATPases. Copper binding studies reveal that this domain has a high affinity for copper(I), with a KD of 2.2 * 10^-18 M.
PfCuP-ATPase is a copper ATPase that is involved in copper metabolism in Plasmodium falciparum. The number of metal-binding domains and their functions is only partially understood. I have identified three N-terminal domains of PfCuP-ATPase and pursued biophysical studies to better understand the structural organization of these domains. Domain 3 is the most soluble and relatively resistant to chemical denaturation with guanidine hydrochloride. 15^N-labelled samples were prepared and 1^H-15^N HSQC NMR experiments were performed for four different constructs. NMR resonance assignments and subsequent Rosetta modelling shows domain 3 to have a ferredoxin fold that is similar to the metal binding domains of human Wilson disease protein. Copper binding studies were performed under anaerobic conditions with CuI(CH3CN)4 + as the copper source, and we obtained a KD of 1.35 * 10-18 M. The presence of a two domain construct, like that of human Wilson protein metal-binding domains five and six, yet lacking a metal-binding motif in domain three is intriguing. This finding highlights the structural versatility and stability of the dual ferredoxin fold.
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Kisaka, Javan Kilango, "N-Terminal Domains of Copper ATPases In Plasmodium Falciparum, Dictyostelium Dicsoideum, Human ATP7B and a Chimera of Atox1 with Metal Binding Domain 4four of ATP7B" (2016). Dissertations. 1614.