Expression, purification and structural properties of ABC transporter ABCA4 and its individual domains

Abstract

ABCA4 is a member of the A subfamily of ATP-binding cassette transporters that consists of large integral membrane proteins implicated in inherited human diseases. ABCA4 assists in the clearance of N-retinylidene-phosphatidylethanolamine, a potentially toxic by-product of the visual cycle formed in photoreceptors during light perception. Structural and functional studies of this protein have been hindered by its large size, membrane association, and domain complexity. Although mammalian, insect and bacterial systems have been used for expression of ABCA4 and its individual domains, the structural relevance of resulting proteins to the native transporter has yet to be established. We produced soluble domains of ABCA4 in E. coli and S. cerevisiae and the full-length transporter in HEK293 cells. Electron microscopy and size exclusion chromatography were used to assess the conformational homogeneity and structure of these proteins. We found that isolated ABCA4 domains formed large, heterogeneous oligomers cross-linked with non-specific disulphide bonds. Incomplete folding of cytoplasmic domain 2 was proposed based on fluorescence spectroscopy results. In contrast, full-length human ABCA4 produced in mammalian cells was found structurally equivalent to the native protein obtained from bovine photoreceptors. These findings offer recombinantly expressed full-length ABCA4 as an appropriate object for future detailed structural and functional characterization.

Clinical spectrum, genetic complexity and therapeutic approaches for retinal disease caused by ABCA4 mutations

“…Mammalian ABC transporters are notoriously difficult substrates for determining crystal structure ( Dahl et al, 2004 ). The best published resolution of the native ABCA4 protein and some mutants, is 18 Å ( Tsybovsky et al, 2010 , 2013 ; Tsybovsky and Palczewski, 2014 ). The lack of high-resolution ABCA4 structure makes functional studies challenging, limiting experimental systems to animal models ( Makelainen et al, 2019 ; Molday et al, 2018 ; Molday and Molday RS, 2016 ; Zhang et al, 2015 ) and in vitro assays, including ATP binding, ATPase activity and vesicular transport studies ( Ahn and Molday, 2000 ; Beharry et al, 2004 ; Sun et al, 1999 ).…”

“…The current status of the structure and (biochemical) function of ABCA4 protein is outside of the scope of this review. These aspects have been described in depth in manuscripts from the laboratories of Robert Molday and Krzysztof Palczewski, and we direct the reader to these papers for excellent overviews of the status of structure and function correlations in ABCA4 ( Molday, 2015 ; Tsybovsky et al, 2013 ; Tsybovsky and Palczewski, 2014 ). We will address the functional studies of ABCA4 variants affecting splicing in depth below.…”

Identification of novel pathogenic ABCA4 variants in a Han Chinese family with Stargardt disease

“…In the present study, amino acid alteration of the hydrophilic glycine residue to the hydrophobic tryptophan residue at position 1203 (p.(Gly1203Trp)) may impact the function or tertiary structure. This alteration located in the cytoplasmic domain 1 (although often called nucleotide binding domain 1) probably covers ABCA4 protein binding sites due to hydrophobic residues mainly tending to locate on molecule surfaces [13,33]. Interestingly, a p.(Gly1203Arg) missense mutation has been reported in an Italian patient with STGD1 [34].…”

“…These suggest the functional importance of the hydrophilic glycine residue. However, the change of leucine residue to proline residue at position 2241 (p.(Leu2241Pro)) may impact retinal-activated ATPase activity on account of locating in the cytoplasmic domain 2 (although often called nucleotide binding domain 2) [13,33]. The p.(Leu2241Val) variant (c.6721C>G), a mutation at the same amino acid position, has been reported in a German individual with STGD1 [35].…”

“…The photoreceptor cell-specific ABCA4 protein encoded by the ABCA4 is an important membrane protein composed of two exocytoplasmic domains, two transmembrane domains, and two cytoplasmic domains, which transport all-trans retinal, a by-product of the retinoid cycle, from intra-disc space to photoreceptor cytoplasm to avoid abnormal accumulation of all-trans retinal in photoreceptors outer segments and in RPE cells [13,14]. As an N-retinylidene-phosphatidylethanolamine (NRPE) transporter, the protein is mainly involved in ATP-dependent vitamin A metabolite translocation from a photoreceptor outer segment disc lumen to cytoplasm [3,14].…”