The studies completed here investigate the mechanism of dysfunction of the E506Q and H537A point mutations in the MsbA ABC transporter.
The inability of the MsbA proteins containing the E506Q or H537A point mutations to sustain cell growth is likely due to the protein binding and/or hydrolyzing ATP and closing in vivo and then unable to reopen and thus unable to provide the necessary transport of lipid A for cell survival. Even though hydrolysis is able to slowly occur, as could be concluded from the in vitro ATPase and ATP detection assay results, if the protein does not reopen, it is no longer useful as a transporter. Our data show that the proteins are still able to bind ATP and slowly hydrolyze ATP to ADP and Pi yet become locked into a closed dimer conformation even when the hydrolysis products are no longer present. If ATP can rebind the closed dimer, then it is reasonable that there is space for the nucleotide to dissociate and create an empty, closed homodimer structure. This closed conformation could also explain the low Km values for E506Q and H537A, where ATP is not as free to dissociate as it would be in the open conformation.
The DEER data support the speculation that the protein gets trapped in the ATP-bound or posthydrolysis state with the hydrolysis products trapped in the binding pocket. This is similar to the occluded state model proposed for Pgp E552A/E1197A, which suggests that loose binding of MgATP at both NBDs (in combination with ligand binding) results in dimer closure with only one tightly bound MgATP.41
Thus, in an effort to determine which nucleotide the purified E506Q and H537A mutants contain natively, a luminescent assay that detects the presence of ATP or ADP was carried out on purified E506Q and H537A MsbA proteins and surprisingly showed that there is no nucleotide in the protein upon purification. This indicates that the nucleotide that is trapped in vivo
is lost sometime during the purification process and that the protein continues to be trapped closed even in the absence of the nucleotide that causes the initial trapping. Alternatively, the intrinsic sampling of conformational states in vivo
even in the absence of ligand or hydrolysis maybe sufficient to generate the interactions required to maintain the closed dimer conformation.
Fluorescence data indicate that both E506Q and H537A retain their ability to bind ATP. These data also support the above conclusion that the proteins must not have nucleotide bound prior to the assay or they would not have WT-like affinity for the TNP-ATP. Compared to published binding values for other ATPase dimers (Kd
examples: MalK, 150 μ
MJ0796, 14 μ
HlyB-NBD, 6.5–7.23 μ
OpuA, 0.9 uM43
), the entire WT MsbA transporter has a Kd
for TNP-ATP of 0.32 μ
M. Using a different method to detect binding of TNP-ATP to MsbA, another group reported Kd
values with significantly lower affinity (50 μ
ATPase data show significantly reduced hydrolysis rates for the E506Q and H537A proteins. The values, which range from 1 to 10% of WT for the reporter pairs and 5 and 8% for E506Q and H537A, respectively, are slightly above background levels. However, all but three of the double mutations, as well as E506Q and H537A, have nonzero hydrolysis rates as indicated by the standard errors in the radioactive ATPase assay as well as corroborating nonzero Vmax values from an independent assay for E506Q and H537A. This, plus the ATP detection assay and CW EPR data, further suggests that hydrolysis does occur over time.
Apo states of each of the reporter mutant pairs studied by CW EPR motional analysis tend to reflect a conformation more in line with the MgATP/Vi state for the reporters observed in the c-less background, and of the five conserved NBD motifs studied, all were motionally affected on some level. In the case of the H537A mutation, not only are each of the five conserved motifs affected by the addition of this point mutation, but the mutation is so detrimental to protein when combined with reporter residues that are otherwise active that the S380C–H537A from the Walker A and T541C–H537A reporting for the H-motif mutants could not be purified. Because the Walker A is thought to bind ATP initially, and only upon dimer closure bring the ATP into contact with the C-loop, Walker B, and H-motifs, the fact that the mobility of the spin-labels at all nine reporters is altered in the apo state upon inclusion of E506Q or H537A indicates that either these point mutations are able to alter the conformational state of the entire NBD or the protein is in a closed state upon purification. This latter possibility is backed up by the fact that the DEER data show a distance of 28 Å between the two S423C sites within the E506Q and H537A homodimers even in the apo state.
The identical MgATP and MgATP/Vi spectra for all but one pair studied also suggests that the proteins are already locked closed and not in need of Vi to lock in the hydrolysis products in a closed dimer conformation as it is in the c-less background experiments. The data show that many of the spectra report motional differences between ATP and MgATP states, which could be due to Mg aiding ATP in binding more tightly to the protein, to changes induced by the binding of Mg itself, or to hydrolysis-induced changes in a small amount of the proteins. AMP-PNP experiments also indicated that H537A shows local conformational changes due to slow hydrolysis of ATP.
The results reported here that E506Q can hydrolyze ATP very slowly over time are consistent with the nonzero (i.e., 10%–
15%) activity data reported for HlyB-NBD E631Q12
and PDR5 E1306Q,17
but not similar to the statements reported for E to Q mutants in BmrA, HlyB-NBD, MalK, and MJ0796 that hydrolysis is defective in these proteins.14–16,19
In addition, the rate of hydrolysis obtained for MsbA E506Q is similar to the 4.4 nmol/(mg min) rate observed for the equivalent mutations (E552Q/E1197Q) in Pgp, which is also similar to that observed for Pgp E552A/E1197A (5.0 nmol/(mg min)).45
And, the results reported here that H537A can also fully hydrolyze ATP, but not on the short time scale of the ATPase assay, are only somewhat consistent with the WT-like activity reported for PDR5 H1068A.17
The comprehensive approach applied to the study of MsbA E506Q and H537A has provided data for the first time that unequivocally state that these point mutations are able to hydrolyze multiple ATPs in the E. coli
The similarities in the impaired function of the E506Q and H537A mutations that create altered resting state conformations and reduce the number of changes throughout the hydrolysis cycle suggest that, since the native E506 and H537 residues may interact, a point mutation at either residue results in a similar dysfunctional state caused by the absent interaction. It is remarkable that single point mutations can generate new inter-molecular interactions within a large protein structure that are unable to be broken even in the absence of nucleotide.