Reading Between the ABCs: Intrinsic Disorder and Evolutionary Dynamics of Non-Canonical Regions in ABC Transporters

ATP-binding cassette (ABC) transporters constitute one of the largest membrane pro-tein superfamilies, yet the structural and evolutionary properties of their non-domain re-gions remain poorly characterized. To elucidate the diversity of these non-canonical re-gions across evolutionary lineages, we analyzed intrinsic disorder, site-specific selection, and predicted post-translational modification (PTM) sites across five architectural classes comprising 1,581 prokaryotic and eukaryotic sequences. Linker and flanking regions were consistently more disordered than transmembrane and nucleotide-binding domains across all architectures. Disorder fraction differed significantly among region types after phyloge-netic correction (Pagel's λ ≈ 0.97). Predicted PTM sites are enriched in disordered non-domain segments, with N-linked glycosylation and phosphoserine showing the strongest positive enrichment; 140 sites satisfied a tiered conservation criterion (Mu-siteDeep score ≥ 0.5; cross-species conservancy ≥ 30%), including 40 high-confidence or moderate-confidence sites (conservancy ≥ 50%) as well as novel phosphotyrosine candi-dates in half transporters and NBD-only proteins. Site-specific selection analyses indicated that episodic positive selection was concentrated at inter-domain boundaries, whereas NBD cores were subject to pervasive purifying selection. Together, these findings establish that non-canonical regions of ABC transporters are evolutionarily dynamic and harbor conserved predicted modification sites, supporting their roles as potential regulatory inter-faces rather than passive structural linkers.