Analysis of tryptophan residues in the staphylococcal multidrug transporter QacA reveals long-distance functional associations of residues on opposite sides of the membrane.
Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein.
Hassan
Karl A
KA
School of Biological Sciences, Flinders University, Adelaide, South Australia, Australia, 5001.
Souhani
Talal
T
Skurray
Ronald A
RA
Brown
Melissa H
MH
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001548:Benzalkonium Compounds / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002710:Chlorhexidine / Q000494:pharmacology; D003868:Dequalinium / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium / Q000494:pharmacology; D005452:Fluoresceins / Q000737:chemistry; D007211:Indoles / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010419:Pentamidine / Q000494:pharmacology; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011754:Pyronine / Q000494:pharmacology; D012235:Rhodamines / Q000494:pharmacology; D013210:Staphylococcus / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D001548:Benzalkonium Compounds; D005452:Fluoresceins; D007211:Indoles; D026901:Membrane Transport Proteins; D012235:Rhodamines; C026188:rhodamine 6G; C068498:qacA protein, Staphylococcus aureus; C007293:DAPI; D010649:Phenylalanine; D010419:Pentamidine; C037041:fluorescein 5-maleimide; D014364:Tryptophan; D003868:Dequalinium; D004996:Ethidium; D000409:Alanine; D002710:Chlorhexidine; D011754:Pyronine