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Split tasks of asymmetric nucleotide‐binding sites in the heterodimeric <scp>ABC</scp> exporter EfrCD
- Source :
- The FEBS Journal. 284:1672-1687
- Publication Year :
- 2017
- Publisher :
- Wiley, 2017.
-
Abstract
- Many heterodimeric ATP-binding cassette (ABC) exporters evolved asymmetric ATP-binding sites containing a degenerate site incapable of ATP hydrolysis due to noncanonical substitutions in conserved sequence motifs. Recent studies revealed that nucleotide binding to the degenerate site stabilizes contacts between the nucleotide-binding domains (NBDs) of the inward-facing transporter and regulates ATP hydrolysis at the consensus site via allosteric coupling mediated by the D-loops. However, it is unclear whether nucleotide binding to the degenerate site is strictly required for substrate transport. In this study, we examined the functional consequences of a systematic set of mutations introduced at the degenerate and consensus site of the multidrug efflux pump EfrCD of Enterococcus faecalis. Mutating motifs which differ among the two ATP-binding sites (Walker B, switch loop, and ABC signature) or which are involved in interdomain communication (D-loop and Q-loop) led to asymmetric results in the functional assays and were better tolerated at the degenerate site. This highlights the importance of the degenerate site to allosterically regulate the events at the consensus site. Mutating invariant motifs involved in ATP binding and NBD closure (A-loop and Walker A) resulted in equally reduced transport activities, regardless at which ATP-binding site they were introduced. In contrast to previously investigated heterodimeric ABC exporters, mutation of the degenerate site Walker A lysine completely inactivated ATPase activity and substrate transport, indicating that ATP binding to the degenerate site is essential for EfrCD. This study provides novel insights into the split tasks of asymmetric ATP-binding sites of heterodimeric ABC exporters.
- Subjects :
- Models, Molecular
0301 basic medicine
Consensus site
Stereochemistry
Amino Acid Motifs
Allosteric regulation
Biological Transport, Active
ATP-binding cassette transporter
medicine.disease_cause
Biochemistry
Conserved sequence
03 medical and health sciences
Adenosine Triphosphate
Allosteric Regulation
Bacterial Proteins
ATP hydrolysis
Consensus Sequence
Enterococcus faecalis
medicine
Nucleotide
Amino Acid Sequence
Molecular Biology
chemistry.chemical_classification
Mutation
Binding Sites
Sequence Homology, Amino Acid
030102 biochemistry & molecular biology
Chemistry
Walker motifs
Cell Biology
Recombinant Proteins
030104 developmental biology
Mutagenesis, Site-Directed
ATP-Binding Cassette Transporters
Dimerization
Sequence Alignment
Protein Binding
Subjects
Details
- ISSN :
- 17424658 and 1742464X
- Volume :
- 284
- Database :
- OpenAIRE
- Journal :
- The FEBS Journal
- Accession number :
- edsair.doi.dedup.....8ec3580748a2c20b86c8af73c7804bda