Functional Impact of a Single Mutation within the Transmembrane Domain of the Multidrug ABC Transporter Pdr5

Abstract

The pleiotropic drug resistance network in budding yeast presents a first line of defense against xenobiotics, which is formed by primary and secondary active membrane transporters. Among these transporters, the ABC transporter Pdr5 is a key component, because it confers resistance against a broad spectrum of such cytotoxic agents. Furthermore, it represents a model system for homologous transporters from pathogenic fungi and has been intensively studied in the past. In addition to other mutational studies, the S1360F mutation of Pdr5 was found to modulate substrate specificity and resistance. Notably, in the S1360F background, the resistance against the immunosuppressant FK506 is drastically increased. We present a detailed analysis of this mutation that is located in the predicted cytosolic part of transmembrane helix 11. Our data demonstrate that kinetic and thermodynamic parameters of the S1360F mutant are similar to those of the wild-type protein, except for FK506-inhibited ATPase activity and the degree of competitive inhibition. In summary, our results indicate that the S1360F mutation within the transmembrane domain interferes drastically with the ability of the nucleotide-binding domains to hydrolyze ATP by interfering with interdomain crosstalk.

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae

“…Thus, their observations indicate that Snq2p and Pdr5p share common substrates, but also have specificities. The relationships between Pdr5p structure and activity have been extensively studied, 20,[28][29][30][31][32] but the structure-function relationships of Snq2p have not been reported. Therefore, it is interesting and important to compare Snq2p and Pdr5p for improving our understanding of ABC transporters.…”

Cowpox Virus Protein CPXV012 Eludes CTLs by Blocking ATP Binding to TAP

“…During the peptide-transport cycle, the TM helices form several conformational intermediates that depend on substrate and ATP binding to cytosolic domains of TAP (22,49). The communication between the TM helices and the cytosolic domains is bidirectional, because the TM helices can also influence substrate and ATP binding at the cytosolic domains (50,51). By binding directly to the TM domains of TAP, CPXV012 may lock TAP in an intermediate conformation, thereby preventing ATP binding to the cytosolic NBDs and interrupting the peptide-transport cycle.…”

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

“…The binding pocket of Pdr5 is a busy place with multiple drug-binding regions (23,25) and intradomain signaling residues (24). The work in this report demonstrates that these alone are insufficient for the successful removal of xenobiotic compounds from the cell.…”

“…Previously reported mutations in Pdr5 with broad hypersensitivity, such as K911A (Walker A), E1036Q, and G312A, exhibited either a strong reduction in steady-state ATPase activity (13,20) or a loss of communication between the transmembrane domains and NBDs causing a large reduction in allosteric inhibition (14,17). This includes the S1360F mutant, which is in the same TMH as S1368A (21,24). The most surprising feature of this mutant, however, was its behavior in a series of R6G transport assays.…”