Abstract
Mutations in ABC subfamily C member 6 (ABCC6) transporter are associated with pseudoxanthoma elasticum (PXE), a disease resulting in ectopic mineralization and affecting multiple tissues. A growing number of mutations have been identified in individuals with PXE. For most of these variants, no mechanistic information is available regarding their role in normal and pathophysiologies. To assess how PXE-associated mutations alter ABCC6 biosynthesis and structure, we biophysically and biochemically evaluated the N-terminal nucleotide-binding domain. A high-resolution X-ray structure of nucleotide-binding domain 1 (NBD1) of human ABCC6 was obtained at 2.3 Å that provided a template on which to evaluate PXE-causing mutations. Biochemical analysis of mutations in this domain indicated that multiple PXE-causing mutations altered its structural properties. Analyses of the full-length protein revealed a strong correlation between the alterations in NBD properties and the processing and expression of ABCC6. These results suggest that a significant fraction of PXE-associated mutations located in NBD1 causes changes in its structural properties and that these mutation-induced alterations directly affect the maturation of the full-length ABCC6 protein.
Highlights
Pseudoxanthoma elasticum (PXE)3 is a disease of ectopic calcification of elastic tissues, affecting the vasculature, gastrointestinal tract, skin, and eye [1]
These include the mammalian ABCC1/MRP1 and ABCC8/SUR1 structures solved by EM, as well as multiple bacterial ATP Binding Cassette (ABC) proteins that have been solved by X-ray crystallography (18 –21)
A more complete view of both the structure and the folding of ABC subfamily C member 6 (ABCC6) may prove useful in understanding the molecular defects associated with mutations and disease development, as well as potential mechanisms that could be leveraged for rational therapeutic development
Summary
The human ABCC6 protein is composed of five distinct domains: three transmembrane domains and two cytosolic, nucleotide-binding domains (Fig. 1). As with other members of the ABCC subfamily, the extended Walker B sequences in NBD1 of ABCC1/MRP1 and ABCC8/SUR1 deviate from the canonical DE sequence with a substitution of Asp for the ultimate Glu residue [22]. This domain contains the canonical Walker A and B sequences as well as the H- and D-loops (Fig. 3B) This core region of the NBD is structurally highly conserved across the NBD proteins. This domain is highly similar to those of ABCC1/MRP1 and ABCC8/SUR1 when comparing both local structure and sequence similarity [20, 21]. This subdomain contains the LSGGQ signature sequence and the structurally diverse region
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