title: 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.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden.
authors.forename: Ranjeet
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden.
authors.forename: Candan
authors.initials: C
authors.lastname: Ariöz
authors.affiliation: Department of Chemistry, Umeå University, 90187, Umeå, Sweden.
authors.forename: Yaozong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden.
authors.forename: Niklas
authors.initials: N
authors.lastname: Bosaeus
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden.
authors.forename: Sandra
authors.initials: S
authors.lastname: Rocha
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden. Pernilla.wittung@chalmers.se.
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D055592:Biophysical Phenomena; D003300:Copper / Q000737:chemistry; D000073840:Copper-Transporting ATPases / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics; D006527:Hepatolenticular Degeneration / Q000235:genetics* / Q000473:pathology; D006801:Humans; D017354:Point Mutation; D000072757:Protein Conformation, beta-Strand / Q000235:genetics; D000072417:Protein Domains; D055550:Protein Stability; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C066141:MBD1 protein, human; C553236:MBD6 protein, human; D014157:Transcription Factors; D003300:Copper; D004706:Endodeoxyribonucleases; C118712:MBD4 protein, human; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases
keywords: ATP7B; Circular dichroism; Metal-binding domain; Molecular dynamics; Thermal stability; Wilson disease