Evolutionary origin and sequence signatures of the heterodimeric ABCG5/ABCG8 transporter

Abstract

ATP-binding cassette (ABC) systems, characterized by ABC-type nucleotide-binding domains (NBDs), play crucial roles in various aspects of human physiology. Human ABCG5 and ABCG8 form a heterodimeric transporter that functions in the efflux of sterols. We used sequence similarity search, multiple sequence alignment, phylogenetic analysis, and structure comparison to study the evolutionary origin and sequence signatures of ABCG5 and ABCG8. Orthologs of ABCG5 and ABCG8, supported by phylogenetic analysis and signature residues, were identified in bilaterian animals, Filasterea, Fungi, and Amoebozoa. Such a phylogenetic distribution suggests that ABCG5 and ABCG8 could have originated in the last common ancestor of Amorphea (the unikonts), the eukaryotic group including Amoebozoa and Opisthokonta. ABCG5 and ABCG8 were missing in genomes of various lineages such as snakes, jawless vertebrates, non-vertebrate chordates, echinoderms, and basal metazoan groups. Amino-acid changes in key positions in ABCG8 Walker A motif and/or ABCG5 C-loop were observed in most tetrapod organisms, likely resulted in the loss of ATPase activity at one nucleotide-binding site. ABCG5 and ABCG8 in Ecdysozoa (such as insects) exhibit elevated evolutionary rates and accumulate various changes in their NBD functional motifs. Alignment inspection revealed several residue positions that show different amino-acid usages in ABCG5/ABCG8 compared to other ABCG subfamily proteins. These residues were mapped to the structural cores of transmembrane domains (TMDs), the NBD-TMD interface, and the interface between TMDs. They serve as sequence signatures to differentiate ABCG5/ABCG8 from other ABCG subfamily proteins, and some of them may contribute to substrate specificity of the ABCG5/ABCG8 transporter.

Keywords: ABC systems; ABC transporters; ABCG5; ABCG8; sequence signatures; sterol efflux.

FIGURE 1

Multiple sequence alignment of sequence motifs in NBDs of the ABCG subfamily proteins. Consensuses of NBD sequence motifs are marked on top of the alignment. Conserved amino acids in these motifs are marked by red bold letters. Substitutions in these positions are marked by bold, black and underlined letters. Each protein is denoted by a two‐letter species abbreviation code (hs, Homo sapiens; cg, C. gigas; dm, D. melanogaster; is, I. scapularis; sc, S. cerevisiae; pv, P. violaceum; at, A. thaliana) and its NCBI accession number. Available common gene names are placed after the accession numbers for human, fruit fly, budding yeast, and A. thaliala. N‐terminal and C‐terminal NBDs of full transporters are denoted by “_N” and “_C” after the common names, respectively. Names of possible inactive NBDs due to substitutions in motifs are underlined. Organism names are colored as follows: magenta: metazoan; black: Filasterea; orange: fungi; blue: amoebozoan; green: green plants. The position showing different amino acid composition in ABCG5/ABCG8 is highlighted in grey background, with conserved amino acids in ABCG5/ABCG8 in bold magenta letters. The amino acid position numbers in human proteins are labeled above (Q206 for ABCG5 and Q226 for ABCG8). Numbers of amino acids in between the blocks are shown in parentheses

FIGURE 2

Multiple sequence alignment showing ABCG5/ABCG8‐signature residues in TMDs of ABCG subfamily proteins. Sequences are named and ordered in the same way as in Figure 1. Small residues (G, A, S, C, T, P) in positions with mainly small residues are colored green. Residues in positions showing differences in amino acid usages of ABCG5 or ABCG8 compared to other ABCG subfamily proteins are highlighted in grey or yellow background. These positions are labeled by their amino acid numbers in human ABCG5 and ABCG8 proteins. The signature residues in ABCG5 and ABCG8 are colored based on their locations in the structure of the human ABCG5/ABCG8 complex: magenta—at the interface of TMD and NBD; cyan—positions in the interface of ABCG5 TMD and ABCG8 TMD; orange—polar residues in the core of the transmembrane helices, yellow background—positions showing size changes in ABCG5 TMD. Ligand‐interacting residues are marked by asterisks above human ABCG5 and ABCG8 (PDB: 7R8B, site 1 residues in green and site 2 residues in red), human ABCG1 (PDB: 7R8D), and human ABCG2 (PDB: 7OJ8)

FIGURE 3

Phylogenetic tree of selected ABCG subfamily proteins. Sequence names are denoted the same way as in Figure 1. Sequences with possible inactive ATPases due to substitutions in conserved motifs are denoted by underlined names and red branches

FIGURE 4

Structural mapping of ABCG5/ABCG8‐signature residues. C‐alpha traces of ABCG5 and ABCG8 are shown in black and gray, respectively. Side chains of ABCG5 and ABCG8 signature residues are shown in spheres, and the residue numbers are labeled for them. Color coding of side‐chain carbon atoms in these residues is consistent with the color coding of these residues in Figures 1 and 2. Sterol molecules in site 1 and site 2 are colored in green and red, respectively