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

Zuben E Sauna,Sherry Supernavage Bohn,Robert Rutledge,Michael P Dougherty,Susan Cronin,Leopold May,Di Xia,Suresh V Ambudkar,John Golin

doi:10.1074/jbc.m806446200

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.

Highlights

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins
A central issue with all ATPbinding cassette (ABC) drug transporters is the coupling of ATP hydrolysis and drug transport
We examined residues in the yeast ABC transporter Pdr5 that helped us define an interface that is probably involved in this process

Summary

Relevant genotype

Mat a PDR5 PDR1-3 ura leu MAT␣ PDR1-3 ura his yor pdr snq pdr ycf pdr pdr. Recent studies with P-gp and Yor used site-directed mutagenesis and cross-linking agents to demonstrate the proximity of ICL2 and the Q-loop [11, 12] These studies show or imply a physical interaction, but none establish that these regions are involved in communication between the TMDs and NBDs. Demonstrating the functional relevance of such interactions remains a challenge. An allosteric inhibitory signal mediated by the Pdr5-specific transport substrate clotrimazole is greatly diminished These deficiencies are significantly alleviated by a suppressor mutation that lies near the Q-loop of NBD1. Our strategy of using suppressor mutations to identify the interacting residues in the TMDs and NBDs has obvious advantages over the use of site-directed mutagenesis This powerful approach can be applied to any mammalian transporter that is expressed in yeast and has a readily observable phenotype

EXPERIMENTAL PROCEDURES

RESULTS

Median fluorescence

Phenotypes of suppressors

DISCUSSION