Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Biochemistry (Impact Factor: 3.02). 04/2011; 50(18):3599-608. DOI: 10.1021/bi101666p
Source: PubMed


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.

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Available from: Candice S Klug, Feb 20, 2015
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    • "Since the range of IF MsbA conformers did not change in response to nucleotide binding (Figure 4E), we present only the fraction of OF particles in subsequent studies to simplify data analysis. First, to capture an ATP-bound, prehydrolysis state, we analyzed several conditions, including MsbA incubations with a nonhydrolyzable ATP analog AMP-PNP, depletion of Mg 2+ , an essential cofactor for ATP hydrolysis, and the use of a catalytically inactive MsbA mutant, E506Q, which permits ATP binding but abolishes ATP hydrolysis (Cooper and Altenberg, 2013; Schultz et al., 2011). The results show that all conditions mimicking the ATP-bound state resulted in an increased population of OF particles compared with the APO state (Figure 5A; Table S1), indicating that the OF state is populated by nucleotide binding. "
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