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Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA. Biochemistry 2011 May 10;50(18):3599-608

Date

04/06/2011

Pubmed ID

21462989

Pubmed Central ID

PMC3128438

DOI

10.1021/bi101666p

Scopus ID

2-s2.0-79955628177 (requires institutional sign-in at Scopus site)   26 Citations

Abstract

MsbA is a member of the ABC transporter superfamily that is specifically found in Gram-negative bacteria and is homologous to proteins involved in both bacterial and human drug resistance. The E506Q and H537A mutations have been introduced and used for crystallization of other members of the ABC transporter protein family, including BmrA and the ATPase domains MalK, HlyB-NBD, and MJ0796, but have not been previously studied in detail or investigated in the MsbA lipid A exporter. We utilized an array of biochemical and EPR spectroscopy approaches to characterize the local and global effects of these nucleotide binding domain mutations on the E. coli MsbA homodimer. The lack of cell viability in an in vivo growth assay confirms that the presence of the E506Q or H537A mutations within MsbA creates a dysfunctional protein. To further investigate the mode of dysfunction, a fluorescent ATP binding assay was used and showed that both mutant proteins maintain their ability to bind ATP, but ATPase assays indicate hydrolysis is severely inhibited by each mutation. EPR spectroscopy data using previously identified and characterized reporter sites within the nucleotide binding domain along with ATP detection assays show that hydrolysis does occur over time in both mutants, though more readily in the H537A protein. DEER spectroscopy demonstrates that both proteins studied are purified in a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide.

Author List

Schultz KM, Merten JA, Klug CS

Authors

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin
Kathryn M. Schultz Research Scientist I in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

ATP-Binding Cassette Transporters
Adenosine Triphosphatases
Adenosine Triphosphate
Bacterial Proteins
Cysteine
Dimerization
Electron Spin Resonance Spectroscopy
Escherichia coli
Hydrolysis
Kinetics
Lipid A
Models, Biological
Mutagenesis, Site-Directed
Mutation
Nucleotides
Protein Binding