Abstract
After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.
Highlights
Copper (Cu) is found in the active sites of many essential proteins that participate in key cellular reactions (Huffman and O’Halloran 2001; Puig and Thiele 2002; Harris 2003)
After the uptake of Cu ions (Ohrvik and Thiele 2014), the Cu chaperone Atox1 transports the metal to the membrane-bound ATP7A and ATP7B (Menke’s and Wilson disease proteins, respectively), two homologous P1B-type ATPases located in the trans-Golgi network
Thermal unfolding experiments of the apo-forms of the variants probed by circular dichroism (CD) at 222 nm demonstrated that the wild-type protein unfolded in a partly reversible process, whereas MBD4D unfolded irreversibly (Figs. 3a, S2)
Summary
Copper (Cu) is found in the active sites of many essential proteins that participate in key cellular reactions (Huffman and O’Halloran 2001; Puig and Thiele 2002; Harris 2003). Because Atox can deliver Cu to the MBDs (Pufahl et al 1997; Wernimont et al 2000; Achila et al 2006; Banci 2006, 2008, 2009a, 2009b), one may speculate that Cu-triggered conformational changes among these domains might initiate the catalytic cycle upon Atox1-mediated Cu delivery (Mondol et al 2016) It remains unclear if the direct path for Cu goes through the MBDs or, if Atox, like its bacterial homolog (Gonzalez-Guerrero and Arguello 2008), delivers Cu directly to a binding site at the membrane-spanning parts of ATP7A/B. It has been shown in vitro that several MBDs can be deleted/mutated without loss of Cu transport activity, Biometals (2017) 30:27–35 but the presence of at least one MBD appears to be required (Forbes et al 1999; Morin et al 2009)
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