Stabilization of Nucleotide Binding Domain Dimers Rescues ABCC6 Mutants Associated with Pseudoxanthoma Elasticum

Abstract

Edited by Norma AllewellABC 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 ϳ200 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. Pseudoxanthoma elasticum (PXE)2 is a multisystem autosomal recessive disease that results in the dystrophic mineralization of elastic tissues putatively due to the loss of one or more circulatory factors (1, 2). The mineralization and subsequent degradation of elastic fibers impacts multiple organs, including the vasculature, eyes, skin, and gastrointestinal tract (3). Patients suffer from premature arteriosclerosis, reduced peripheral circulation, loss of central vision, loss of skin tone, and bleeding in the digestive tract (4). Recent work suggests that a loss of circulating pyrophosphates, resulting from altered nucleotide secretion and conversion by nucleotide pyrophosphatases in the circulatory system, is associated with ectopic mineralization (5, 6). At present, only symptomatic treatments exist for PXE (4, 7).PXE is caused by mutations in the ATP-binding cassette transporter C6 (ABCC6), a member of the multidrug resistant group of ABC proteins (1, 8 -10). ABC transporters are active transporters that couple the energy of ATP hydrolysis to the directional transport of solutes (11). Although multiple putative substrates have been identified for ABCC6, the PXE-specific solute(s) and the mechanisms of ABCC6 functional regulation have not been fully elucidated (5, 9, 12). ABCC6 is composed of five domains: three transmembrane domains (TMD0/1/2) and two cytosolic nucleotide binding domains (NBD1/2) (13, 14). The core TMDs (TMD1/2) facilitate solute movement across the membrane, whereas the NBDs provide the energy for transport through nucleotide binding and hydrolysis. The NBDs heterodimerize in response to binding two ATP molecules within the conserved domain-domain interface. This dimerization induces conformational changes in the NBDs that propagate through the TMDs (15-17). ATP hydrolysis putatively reverses these conformational changes, thereby promoting transport cycling and multiple rounds of solute efflux. The NBDs are physically coupled to the TMDs through extensions of the TMD helices and intracellular loops, and these interactions are disrupted in multiple ABC transporter disease states (17-20). It is not known how the N-terminal TMD (TMD0...

Structural analysis reveals pathomechanisms associated with pseudoxanthoma elasticum–causing mutations in the ABCC6 transporter

“…These mutants showed little to no fully glycosylated species and steady-state expression was reduced to less than 10%, when compared with the WT protein. The reduction in steady-state protein and lack of fully glycosylated species was consistent with ER-associated degradation, as previously described for processing mutants in ABCC6 (15,28). The R760W, V810M, and T811M mutants produced small amounts of fully glycosylated protein, although steady-state expression was markedly reduced compared with WT.…”

“…This could suggest a direct role for nucleotide during crystallization. Alternatively, the nucleotide may stabilize the free pool of NBD1 and prevent aggregation, as has been demonstrated for other NBDs, thereby indirectly facilitating crystallization (15,30,31). The adverse effects of the D777N, reported here, and D778N, previously reported, in the Walker B sequence suggest that Mg-ATP binding may be critical for the folding and/or structural stability of ABCC6 NBD1 (15).…”

“…Although many PXE-associated alleles are yet to be characterized, previous studies have implicated protein misfolding as a potential molecular mechanism underlying this disease (15)(16)(17)28). Recent advances in drug development efforts provide evidence that small molecule corrections of these defects can provide therapeutic relief for patients (29).…”

“…The exact molecular pathologies associated with the majority of these mutations are not known. Recent studies suggest that alterations to the biosynthesis and trafficking of ABCC6 influence a subset of PXE-causing mutations, although the detailed mechanisms of how these mutations alter ABCC6 structure is unknown (5,15,16). In addition, recent studies suggest that ABCC6 misfolding can be partially corrected, suggesting that reversion of these basic defects could be leveraged for therapeutic development (17).…”

Insights into Pathomechanisms and Treatment Development in Heritable Ectopic Mineralization Disorders: Summary of the PXE International Biennial Research Symposium—2016

“…Furthermore, systematic evaluation of a large library of ABCC6 mutations has identified mutations leading to altered protein folding and altered protein function. Preliminary data have also demonstrated that stabilization of mutant ABCC6, via specific domain-domain interactions, can facilitate proper trafficking and function of the mutant proteins, providing a mechanism for stabilization of the protein and rescue of the phenotype due to disease-causing mutations (Ran and Thibodeau, 2017). …”