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