Molecular Modeling of the Transmembrane Regions and Nucleotide Binding Domains in the ABCA4 Transporter

Abstract

Purpose: : Mutations affecting the ABCA4 transporter protein are known to cause autosomal recessive retinal degenerative diseases, including Stargardt macular degeneration. More than 50% of pathogenic missense mutations causing Stargardt disease are associated with changes in the transmembrane regions (TM’s) and nucleotide binding domains (NBD’s) of ABCA4. Here we model a structure for TM’s and NBD’s and examine pathogenic mutations that cause Stargardt macular degeneration for their effect on the structure and function of ABCA4.

Methods: : The structure of TM’s and NBD’s of ABCA4 was generated by homology modeling using a bacterial lipid flippase MsbA as a template. The open and closed conformers were both minimized and then placed in an 80x80 1-[2H₃₁] palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine membrane with the most hydrophobic regions of the ABCA4 surface buried in the hydrocarbon core of the membrane. The ATP/ADP binding site was located using known ATP binding motifs. NRPE structure was generated using atomic structures of retinal and phosphatidylethanolamine. In order to analyze the structural effects of mutations, the accessibility of each residue has been determined. The residues with an accessibility of less than 40 angstrom² was considered buried. The location and size of the cavities for the open and closed conformers were obtained using PoreWalker.

Results: : According to our modeling, ATP dephosphorylation drives the ABCA4 conformation from the closed structure to an open state. In contrast to the closed conformer with a compact structure, the open conformer is a "V-shaped" structure with a large "V-shaped" opening filled with aqueous solution. The conformational change causes the NRPE flipping to the leaflet of the membrane. In BIOBASE, 156 mutations affect the TM’s or NBD’s and are associated with Stargardt disease. The mutations were classified into 4 groups: 34% affect the NRPE transport process, 31% affect the protein surface, 34% affect protein stability, and 16% affect chain dimerization.

Conclusions: : Knowledge of the structure, transport mechanism, and effect of mutations on ABCA4 confirms previous data that ABCA4 functions as a lipid flippase, flipping NRPE across the photoreceptor outer segment disc membrane, and might provide a deeper understanding of the pathology of Stargardt macular degeneration.

Keywords: protein structure/function • computational modeling • proteins encoded by disease genes