title: Stabilization of Nucleotide Binding Domain Dimers Rescues ABCC6 Mutants Associated with Pseudoxanthoma Elasticum.
abstract: ABC transporters are polytopic membrane proteins that utilize ATP binding and hydrolysis to facilitate transport across biological membranes. Forty-eight human ABC transporters have been identified in the genome, and the majority of these are linked to heritable disease. Mutations in the ABCC6 (ATP binding cassette transporter C6) ABC transporter are associated with pseudoxanthoma elasticum, a disease of altered elastic properties in multiple tissues. Although ∼200 mutations have been identified in pseudoxanthoma elasticum patients, the underlying structural defects associated with the majority of these are poorly understood. To evaluate the structural consequences of these missense mutations, a combination of biophysical and cell biological approaches were applied to evaluate the local and global folding and assembly of the ABCC6 protein. Structural and bioinformatic analyses suggested that a cluster of mutations, representing roughly 20% of the patient population with identified missense mutations, are located in the interface between the transmembrane domain and the C-terminal nucleotide binding domain. Biochemical and cell biological analyses demonstrate these mutations influence multiple steps in the biosynthetic pathway, minimally altering local domain structure but adversely impacting ABCC6 assembly and trafficking. The differential impacts on local and global protein structure are consistent with hierarchical folding and assembly of ABCC6. Stabilization of specific domain-domain interactions via targeted amino acid substitution in the catalytic site of the C-terminal nucleotide binding domain restored proper protein trafficking and cell surface localization of multiple biosynthetic mutants. This rescue provides a specific mechanism by which chemical chaperones could be developed for the correction of ABCC6 biosynthetic defects.
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15219.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Ran
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15219. Electronic address: thibodea@pitt.edu.
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Thibodeau
mesh_terms: D019943:Amino Acid Substitution; D057809:HEK293 Cells; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D000072417:Protein Domains; D055503:Protein Multimerization; D055550:Protein Stability; D021381:Protein Transport / Q000235:genetics; D011561:Pseudoxanthoma Elasticum / Q000235:genetics / Q000378:metabolism
chemical_list: D027425:ATP-Binding Cassette, Sub-Family C Proteins; C102029:ABCC6 protein, human
keywords: ABC transporter; abcc6; biosynthesis; calcification; protein stability; protein structure; pseudoxanthoma elasticum