Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics

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.

Keywords: ATP7B; Circular dichroism; Metal-binding domain; Molecular dynamics; Thermal stability; Wilson disease.

Fig. 1

Amino acid sequence and 3D structure of MBD4 variants. The color scheme of the primary sequences follows that of 3D structure; secondary structures are differently colored. Gly386 is presented as a sphere and the Cu-binding Cys are shown in stick representation

Fig. 2

CD spectra of MBD4D (red), MBD4V (blue), and MBD4 wild-type (black): a without Cu, and b in the presence of Cu in a 1:1 molar ratio of metal to protein

Fig. 3

Thermal unfolding profiles of MBD4D (red), MBD4V (blue), and MBD4 (black). without Cu (a) and in the presence of Cu in a 1:1 molar ratio (b) probed by CD at 222 nm. Circles indicate estimated midpoints of the unfolding transitions (the first transition for MBD4V)

Fig. 4

CD spectra of MBD4V at 10 °C (blue), 48 °C (purple), 98 °C (red), and, upon re-cooling of heated sample, at 5 °C (yellow)

Fig. 5

Structural deviation and fluctuation of MDB4 proteins. a Backbone RMSD profiles of MBD4, MBD4V and MBD4D (black, blue and red, respectively). The smoothed curves are shown together with their corresponding RMSD curves. b Heavy atom RMSF profiles with the same color scheme as in (a)

Fig. 6

Covariance matrices of MBD4 (a), MBD4V (b) and MBD4D (c) with the X and Y axes reporting on residue ID. The color scale changes from blue (−0.6 and smaller correlation value) for anti-correlation to green (zero) for non-correlation and to red (0.6 and greater correlation value) for correlation between a pair of residues