Coevolving Amino Acid Positions in Exporter-Type ABC Proteins

Abstract

Residue-residue interactions define the protein fold, and their dynamic interplay mediates conformational rearrangements between global states, such as the outward- or inward-facing conformations of transporters. These physical interactions constrain sequence evolution by coupling the pattern of amino acid substitution at interacting positions (coevolution). Thus, identification of coevolving positions can provide structural and mechanistic insights at the resolution of single residues. Here we identified coevolving positions in the OAD and DPL families of the ABC superfamily. These families harbor exporters involved in multidrug resistance like MDR1/Pgp (DPL) and MRPs (OAD), as well as the CFTR chloride channel (OAD) linked to cystic fibrosis. We generated multiple sequence alignments separately for OADs and DPLs, and analyzed them with three different statistical methods. The three methods yielded somewhat different results likely due to their limited accuracy and differences in their assumptions about mechanisms of coevolution. Nonetheless, the results are validated by three lines of structural evidence, all supporting the hypothesis that direct physical interactions play a major role in coevolution. First, coevolution statistics were significantly linked to spatial distance in a 3D structural model. Second, the methods agreed better if only contacting positions were considered. Third, coevolving pairs were separated in sequences according to the periodicity of alpha helices and beta sheets. We present sets of coevolving pairs that link different transmembrane helices, or that link the coupling helices to the ATP-binding cassettes. Our findings provide specific, testable hypotheses for mutational and crosslinking studies on the detailed transport mechanisms of clinically relevant ABC proteins such as those underlying cystic fibrosis and multidrug resistance. [Supported by DK 51767]