Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5

Abstract

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K m(ATP) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.Multidrug transporters, including members of the ATPbinding cassette (ABC) 2 family, show unusual flexibility toward their substrate cargo. They efflux structurally diverse xenobiotic compounds and confer broad-spectrum hyperresistance when they are overexpressed, a property that impedes chemotherapeutic treatment of pathogens and cancer. Several fungi, including the clinically relevant human pathogenic species Candida albicans and Cryptococcus neoformans, contain major multidrug transporters that are close homologues to the Saccharomyces cerevisiae transporter Pdr5 (1). It is well established that many clinical isolates of C. albicans overproduce the Pdr5 homologue Cdr1 (2). Multidrug-resistant fungi are an increasing problem in the treatment of immunocompromised patients with AIDS and cancer (3).The Pdr5 subfamily of multidrug transporters shows significant differences in molecular architecture from their mammalian counterparts such as P-glycoprotein (P-gp) or Mrp1. The nucleotide-binding domains (NBDs) of these important fungal transporters precede the transmembrane domains (TMDs) and thus their orientation is the reverse of P-gps and Mrp1s. Furthermore, the Walker A, B, and signature motifs are degenerate when compared with nonfungal ABC counterparts. For example, a cysteine replaces a lysine in Walker A of the N-...

Evidence for a Molecular Diode-based Mechanism in a Multispecific ATP-binding cassette (ABC) Exporter

“…We introduced the mutant plasmid DNA into R-1 with a Sigma-Aldrich yeast transformation kit. Genetic testing confirmed that the construct was correctly inserted (17).…”

“…Assay of ATPase Activity-We measured Pdr5-specific ATPase activity as described previously (12) for 8 min at 35°C with 12 g of purified PM vesicle protein derived from cells bearing two copies of either WT or mutant PDR5 genes (17). We verified all ATPase results by carrying out assays with at least two independent PM vesicle preparations/strain.…”

“…We used strains containing two copies of either WT or a mutant PDR5 gene to make purified PM vesicles for ATPase and IAAP photoaffinitylabeling experiments. The rationale for using and the method for generating these strains are described elsewhere (17). We used single-copy strains for all other experiments, including most of the whole-cell R6G transport studies.…”

“…This strain offers numerous other advantages for genetic and biochemical analyses, which are described in detail elsewhere (17,18). We cultured the strains at 30°C.…”

“…We cultured the strains at 30°C. We used the pSS607-integrating plasmid (12) for site-directed mutagenesis, as described previously (17). This plasmid has a WT PDR5 gene under the transcriptional control of its own upstream region as well as a URA3-selectable marker.…”