Cdr1p highlights the role of the non-hydrolytic ATP-binding site in driving drug translocation in asymmetric ABC pumps

https://doi.org/10.1016/j.bbamem.2019.183131Get rights and content
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Highlights

  • Function of the non-catalytic NBS in asymmetric ABC transporters is uncertain.

  • Q1005H mutation acts as a suppressor for debilitating mutant, L529A in TMD of Cdr1p.

  • Q1005H is part of the ABC signature sequence in the non-catalytic NBS.

  • Q1005H mutation in the suppressor re-synchronizes the ATPase engine with the TMDs.

Abstract

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of allocrites across membranes. Two shared nucleotide-binding sites (NBS) participate in this cycle. In asymmetric ABC pumps, only one of them hydrolyzes ATP, and the functional role of the other remains unclear. Using a drug-based selection strategy on the transport-deficient mutant L529A in the transmembrane domain of the Candida albicans pump Cdr1p; we identified a spontaneous secondary mutation restoring drug-translocation. The compensatory mutation Q1005H was mapped 60 Å away, precisely in the ABC signature sequence of the non-hydrolytic NBS. The same was observed in the homolog Cdr2p. Both the mutant and suppressor proteins remained ATPase active, but remarkably, the single Q1005H mutant displayed a two-fold reduced ATPase activity and a two-fold increased drug-resistance as compared to the wild-type protein, pointing at a direct control of the non-hydrolytic NBS in substrate-translocation through ATP binding in asymmetric ABC pumps.

Abbreviations

ABC
ATP-binding cassette
ANI
anisomycin
Cdr1p
Candida drug resistance 1 protein
Cdr2p
Candida drug resistance 2 protein
CFTR
cystic fibrosis transmembrane conductance regulator
CHX
cycloheximide
CnH
connecting helix
CpH
coupling helix
CTZ
clotrimazole
DMSO
dimethyl sulfoxide
ECL
extracellular loop
ICL
intracellular loop
ITR
itraconazole
KTZ
ketoconazole
MCZ
miconazole
MDR
multidrug resistance
MRP1
Multidrug Resistance Protein 1
NR
Nile red
NBD
nucleotide-binding domain
NBS
Nucleotide Binding Site
OM
oligomycin
Pdr5p
Pleiotropic drug resistance 5 protein
PBS
phosphate-buffered saline
PM
plasma membrane
PMSF
phenylmethanesulfonyl fluoride
RB
resuspension buffer
R6G
rhodamine 6G
R123
rhodamine 123
TAP
transporter associated with antigen processing
TMD
transmembrane domain
TMH
transmembrane helix
TLCK
p-tosyl-l-lysine chloromethyl ketone
TPCK
tosyl phenylalanyl chloromethyl ketone
TCSPC
time-correlated single photon counting
VOR
voriconazole
WT
wild-type

Keywords

Antifungal drug resistance
Candida albicans
ABC transporter
ABC signature sequence
Non-hydrolytic nucleotide-binding site

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1

Contributed equally.