Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA

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.

Figure 1

Structure of MsbA. (A) Apo MsbA (PDB: 3B5W) and (B) MsbA NBD with ADP/V_i (labeled arrow and colored by element) (3B5Z). Domains studied here are colored: LSGGQ (red), Walker A (orange), Walker B (green), H-motif (H537) (magenta), and Q-motif (purple). The nine reporter sites studied within and surrounding these motifs are indicated by colored spheres, and arrows point to the locations of E506 (immediately follows the Walker B) and H537 (H-motif).

Figure 2

ATP, E506, E506Q, H537, and H537A arrangement in the MalK and HlyB-NBD crystal structures. (A) WT MalK (1Q12), (B) E159Q MalK (2R6G), (C) E631Q HlyB-NBD (2FGK), and (D) H662A HlyB-NBD (2FGJ). When glutamate is changed to glutamine in both structures, the side chain changes orientation to point in the direction of the ATP with potential new contacts with the ATP molecule. When histidine is changed to an alanine, the interaction between histidine and ATP is lost.

Figure 3

In vivo growth assay results for NBD reporters alone (black, ref 22) and with E506Q (gray) and H537A (white). The in vivo growth assay is used to determine the ability of each plasmid-encoded mutant to maintain cell viability at 44 °C, after which the temperature sensitive chromosomal copy of MsbA is inactivated. Error bars represent the standard deviation for experiments run twice in triplicate.

Figure 4

In vitro ATPase activity results. Rates of hydrolysis over 2 min at 37 °C as compared to WT are reported for the NBD reporters, E506Q, H537A, and the NBD reporters with E506Q. Standard deviations for the E506Q and H537A data represent results carried out in triplicate. *Values greater than 120% of WT: S423C (244%), Q485C (146%), V534C (153%), T541C (140%).

Figure 5

Nucleotide detection assay results. (A) BSA (negative control), WT MsbA (positive control), E506Q MsbA, and H537A MsbA (5 μM) were analyzed using a luminescence assay for remaining ATP concentration after incubation with 10 μM ATP and excess Mg for 30 min at room temperature. (B) Additional time points show that the H537A and E506Q proteins hydrolyzed all available ATP after 2 and 6 h incubations, respectively.

Figure 6

X-band CW EPR spectra of MsbA reconstituted into inner membrane lipids at various stages of the hydrolysis cycle. The figure indicates the color coding of each reporter residue alone^– as well as with the E506Q mutation in the apo state, in the presence of ATP, MgATP/V_i, and MgATP.

Figure 7

X-band CW EPR spectra of MsbA reconstituted into inner membrane lipids at various stages of the hydrolysis cycle. The figure indicates the color coding of each reporter residue alone^– as well as with the H537A mutation in the apo state, in the presence of ATP, MgATP/V_i, and MgATP. The S380C–H537A and T541C–H537A proteins were not able to be purified.

Figure 8

X-band CW EPR spectral comparison of the MgAMP-PNP-and MgATP-bound (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), MgATP (purple), and MgAMP-PNP (orange).

Figure 9

X-band background-corrected DEER evolutions (left) for S423C (black), S423C–E506Q (gray), and S423C–H537A (white) and the corresponding distance distribution (right) following protein purification in the absence of added ligand. The same concentration of protein was used in each of the samples; thus, the spectral overlays represent a direct comparison of spin populations in the closed dimer conformation. All three samples gave a distance distribution centered on 28 Å.