Spontaneous suppressors against debilitating transmembrane mutants of Ca Mdr1 disclose novel interdomain communication via Signature motifs of the Major Facilitator Superfamily

The Major Facilitator Superfamily (MFS) includes multiple families of proteins operating as uniporters, symporters and antiporters for a wide spectrum of substrates. Among them, the multidrug resistance-1 drug:H ⁺ antiporter Ca Mdr1 from Candida albicans is responsible for the efflux of structurally-diverse antifungals. MFS share a common fold of 12-14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS show a high primary structure diversity and few conserved Signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously rescuing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing Ca Mdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127A _TMH1 ->G140D _TMH1 ) or 3-TMHs repeat (F216A _TMH4 ->G260A _TMH5 ), at the hinge of 3-TMHs repeats tandems (R184A _TMH3 ->D235H _TMH4 , L480A _TMH10 ->A435T _TMH9 ), and finally between the N- and C-domains (G230A _TMH4 ->P528H _TMH12 ). Remarkably, most of these mutants belongs to the different Signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS. Results point also to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.