*2.1. Structure*

The general structure of eukaryotic ABC transporters is a four functional unit organization, comprising two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). NBDs bind and hydrolyze ATP to trigger conformational changes in the TMDs, resulting in unidirectional transport across the membrane [1]. Human peroxisomal ABC transporters have a half-transporter structure, with only one TMD and one NBD [16]. In 2017, we proposed a structural model of human ABCD1 based on the crystal structure of the mitochondrial ABC transporter ABCB10, which shows not only the putative structure of ABCD1 in a membrane context but also the complex intricacy of α-helices that constitute the whole transmembrane domain (Figure 2) [17]. Therefore, peroxisomal ABC half-transporters need to homo- or heterodimerize in the peroxisomal membrane in order to constitute a full, active transporter [18,19]. α

**Figure 2.** Structural model of human ABCD1 (reprinted from [17]). (**A**) Ribbon representation of the ABCD1 monomer. TMD helices are numbered from 1 to 6 and rainbow colored from dark blue to red. NBD is in light grey, and intracellular loops (ICL) 1 and 2 are indicated. (**B**) Ribbon representation of the ABCD1 homodimer with the two subunits respectively colored in dark blue and yellow.

Data shows that ABCD1, ABCD2, and ABCD3 are able to interact as homodimers or heterodimers [20–22], although both ABCD1 and ABCD3 are mainly found as homodimers in mammalian peroxisomal membranes [23–25]. Moreover, ABCD1 and ABCD2 homodimers are functional [26,27]. However, the fact that nonfunctional ABCD2 has a transdominant negative effect on ABCD1 [20] suggests that heterodimers of ABCD1 and ABCD2 are also functional and can exist within cells and tissues expressing both proteins. Besides, chimeric proteins consisting of homo- and heterodimers of ABCD1 and ABCD2 are functionally active [19]. Concerning ABCD3, although homodimers and heterodimers with ABCD1 and ABCD2 have been described [22,23,25,28,29], no data is available about the functional value of the ABCD3 dimers. Surprisingly, ABCD1 and ABCD3 were found in different detergent-resistant microdomains [29], implying that these proteins have a different environment in the peroxisomal lipid bilayer, questioning the biological relevance of the ABCD1 and ABCD3 heterodimers. Additionally, native PAGE experiments concerning complex oligomerization confirm that ABCD1 and ABCD2 exist predominantly as homo-tetramers, although both homo- and hetero-tetramers are present [28]. Therefore, we cannot rule out the possibility that hetero-interaction between ABCD1 and ABCD2 occurs in hetero-tetramers composed of two distinct homodimers rather than in complexes composed of two heterodimers. Finally, it remains unclear whether the oligomerization of peroxisomal ABC transporters has any influence on substrate specificity.
