Date of Award
Doctor of Philosophy
Dr. David Huffman
Dr. Blair Szymczyna
Dr. Gellert Mezei
Dr. Douglas Coulter
Wilson disease protein, metal-binding domains
Wilson disease protein (WLNP) is a P1b-type ATPase crucial for maintaining copper homeostasis in humans. Mutations in this protein result in the autosomal recessive disorder Wilson disease, a condition characterized by copper accumulation in the liver and brain. WLNP provides copper for incorporation into cuproproteins and exports excess copper into the bile for excretion. There are six metal-binding domains (MBDs) in WLNP, found within the first 650 amino acids of this 1,465 amino acid protein. Though each MBD has a different amino acid sequence, all MBDs possess a similar ferredoxin fold with a conserved hydrophobic core and a MXCXXC metal-binding motif.
The manner in which the six MBDs communicate with each other and how they affect other cytosolic-facing domains of WLNP is not understood. There is a long linker between the fourth and fifth MBDs that provides spatial separation between the first four and the last two MBDs. To better understand how the first four MBDs function, I pursued a detailed biophysical characterization of these domains. Strikingly, when MBD4 is expressed by itself, it is highly resistant to both chemical and thermal denaturation: 50% of its structure is retained in 5.9 M guanidine hydrochloride (GuHCl) and it has a melting temperature of 78˚C. In contrast, when MBDs1-3 are expressed together as a single protein, 50% of its structure is retained at 2.3 M GuHCl and the melting temperature is 58˚C. Furthermore, the unusual stability of MBD4 is preserved when it is expressed in a protein construct that contains all four MBDs (MBDs1-4). In MBDs1-4, denaturation by GuHCl produced a double sigmoidal curve in which the second sigmoid correlated with that of MBD4 while the first one correlated with MBDs1-3. MBD4 also influenced the thermal denaturation of MBDs1-4, albeit in a more complicated manner. MBDs1-4 did not display a thermal unfolding transition and instead underwent a structural rearrangement that resulted in soluble aggregation. Though the extreme conditions that MBD4 can withstand are never experienced in vivo, the enhanced stability of this domain may play a role in its ability to serve as a primary target of copper acquisition.
Hinz, Alia V.H., "Biophysical Characterization of the First Four Metal-Binding Domains of Human Wilson Disease Protein" (2014). Dissertations. 280.