W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans

International audience; Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Despite extensive investigations, function of the deviant ATP-binding site remains unclear. Here, we report suppressor analysis on a drug-susceptible transmembrane domain mutant, V532D, which identified a suppressor mutation mapping very close to the D-loop in the atypical ATP-binding site. The suppressor mutation, W1038S, restored drug resistance of the V532D mutant, albeit selectively. Both the mutant and the suppressor showed a striking reduction in ATPase levels. The ketoconazole-sensitive ATPase activity of V532D-W1038S revealed modification of the inter-domain signaling pattern. W1038 and D-loop residues of NBD2 have been earlier highlighted in suppressor screen for intracellular loop mutants as well. This also implies their relevance in inter-domain communication. Further, alanine substitution of the highly conserved aspartate (D1033A) in D-loop of NBD2 rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity pointing towards an important role of this region in coupling ATPase activity to substrate transport. Therefore, we propose W1038 as a crucial determinant of signaling between the domains.