Structure-function analysis of ABCA4 membrane transporter: development of a soluble model system
Date
2024
Authors
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Publisher
University of Delaware
Abstract
The fourth member of ABCA subfamily transporter protein, ABCA4, is highly expressed and localized in the rod and cone outer segment discs of photoreceptor cells. It plays a pivotal role in the visual cycle by translocating its physiological substrates (retinoid by-products such as N-retinylidene-phosphatidylethanolamine (N-Ret-PE)) across the disc membrane for further recycling in the retinal pigment epithelium (RPE). Dysfunctions and mutations in the ABCA4 gene lead to various inherited visual diseases such as Stargardt disease (STGD1), cone-rod dystrophy, retinitis pigmentosa, and fundus flavimicalatus. Over 3,000 variants have been identified and linked to the ABCA4 gene. Still, half of these reported variants lack information as to what extent they can affect the molecular/biochemical properties of ABCA4. The large size and the nature of the twelve transmembrane domains (12-TMα-H) of ABCA4 are significant reasons for the slow progress in characterizing ABCA4 genetic variants in vitro. ☐ In this dissertation, I tested the hypothesis of creating a soluble analog of the ABCA4, which would facilitate the study of this integral transporter protein. We aim to obtain the soluble form of ABCA4 from the cytoplasmic extract without the need to reconstitute it into liposomes but retain the overall structure and function of the protein. The foundation of our membrane solubilization approach was based on the QTY code, which utilizes the substitution of hydrophobic amino acids (leucine, isoleucine, valine, and phenylalanine (LIVF)) in membrane-spanning helices with hydrophilic amino acids (glutamine, threonine, and tyrosine (QTY)) possessing electron density map. Our results indicated that modifications of QTY were necessary. These included (i) narrowing down the selected hydrophobic residue (LIVF) from substitutions to exclusively the lipid- exposed residues in the 12-TMα-H, (ii) eliminating clinically reported pathogenic variants from the QTY substitution, and (iii) employing the Asn amino acid with the Gln to substitute the Leu residue based on bioinformatics pathogenicity prediction software. The modified approach, which resulted in a solubilized transporter (ABCA4S), was designated as the QNTY approach to reflect the incorporation of Asn in the approach. Using this approach, I have developed a novel system for efficient expression, solubilization, and characterization of the ABCAS and its disease-associated variants in the baculovirus expression system. Our key findings from designing the soluble construct, ABCA4S, indicate that eliminating deleterious mutation from the QTY substitution played a significant role in preventing the lack of expression of the protein. ABCA4S protein was successfully expressed in high yield utilizing a baculovirus expression system and attained from the cytosolic extract. ☐ Structural and functional analysis of ABCA4S were compared to the native ABCA4N. Computational analysis using molecular modeling AlphaFold-2 compared the structures of the soluble ABCA4S and the native ABCA4N. The superimposition of the structures showed a close alignment and low RMSD values. Functional analysis of retinal stimulated ATPase demonstrated that ABCA4S exhibited comparable activity as the native ABCA4N. We have utilized the platform to analyze selected disease-associated variants and found their effects on retinal stimulated ATPase were comparable to that previously reported in the literature. These results suggest that QNTY presented here can be applied to other large transporter proteins and may aid in the functional assessment of clinically relevant genetic variants and protein function.
Description
Keywords
Protein function, Genetic variants, Photoreceptor cells, Amino acids, Baculovirus expression