ABSTRACT
Wilson protein (WLNP) is a P1b-type ATPase crucial for pumping Cu(I) into the Golgi or alternately, out of the cell. The six approximately 70-80 amino acid (aa) metal-binding domains (MBDs) in the first 650 aa (N-terminus) of this 1,465 aa protein bind Cu(I) avidly. The first four MBDs are proposed to capture Cu(I) initially, then deliver it to MBDs 5 and 6. 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). Steady state and time-resolved fluorescence was used to study the effect of disease-causing mutations in MBD5-6. In the picosecond time regime, both Y532H and V536A relaxed much faster than native protein. This highlights the importance role of residues near the interfacial region between domains 5 and 6. Generally, our results suggest a lack of cooperativity between MBDs. The MBDs operate as cellular ligands for Cu(I), but other cellular components that could participate as biological ligands for Cu(I) or Cu(II) will be discussed.