Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae.
In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrB(HI)). Similar to other RND transporters AcrB(HI) associates with AcrA(HI), the periplasmic membrane fusion protein, and the outer membrane channel TolC(HI). Here, we report that AcrAB(HI) confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC(EC)) protein. To investigate the intracellular dynamics of AcrAB(HI), single cysteine mutations were constructed in AcrB(HI) in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB(HI) and in the presence or absence of AcrA(HI) and TolC(EC). We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrB(HI).
Dastidar
Vishakha
V
Department of Chemistry and Biochemistry, 620 Parrington Oval, Norman, OK 73019, USA.
Mao
Weimin
W
Lomovskaya
Olga
O
Zgurskaya
Helen I
HI
D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006193:Haemophilus influenzae / Q000737:chemistry / Q000187:drug effects*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011487:Protein Conformation / Q000187:drug effects
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; C037041:fluorescein 5-maleimide; D003545:Cysteine