| ATP binding cassette (ABC) transporters are an important and diverse superfamily of proteins that use ATP as a source of energy to transport their ligands. These proteins are found in all organisms and are involved in a variety of human pathologies from cystic fibrosis ( 1-4) to multidrug resistance (5-9). MsbA is a 65 kDa ABC transporter that is responsible for transporting lipid A across the inner membrane of Gram negative bacteria (10). Lipid A is then transported across the periplasm and the outer membrane, where it is found in the outer membrane of Gram negative bacteria. MsbA is an essential protein in Escherichia coli (11), whose knockout causes the toxic accumulation of lipid A in the inner membrane and results in cell death (10). MsbA shares homology with human P-glycoprotein and Lactococcus lactus LmrA, which are shown to be involved in human and bacterial multidrug resistance, respectively (5,8,12-15). Two MsbA monomers, each containing one nucleotide binding domain (NBD) and one transmembrane domain (TMD) come together to form a functional homodimer. MsbA has been crystallized in the absence and presence of nucleotide revealing open (to the cytoplasm), semi-open, and closed conformations (16). The NBDs of ABC transporters, including MsbA, contain five conserved motifs that have been suggested to play important roles in ATP binding and hydrolysis ( 17). The further study of MsbA will provide a better understanding of not only MsbA, but all ABC transporters.;In these studies, the in vivo cell growth, in vitro ATP binding and ATPase activity of WT MsbA were also characterized using biochemical assays. Sites within the NBD were characterized, including nine residues that were chosen from the five conserved motifs mentioned above, using the same biochemical assays, as well as CW and pulsed EPR spectroscopy (18). It was determined that ATP binds to the Walker A motif (residues 378-384) first and interacts with the other conserved motifs only after dimer closure. A number of important residues were identified by loss of growth and ATPase activity after cysteine substitution.;The E506Q mutation, found immediately after the Walker B motif, and H537A, a mutation of the conserved H-motif, have been identified and described previously in other ABC transporters, but the cause of dysfunction had not yet been determined. The E506Q and H537A point mutations were characterized using the same techniques listed above and, with comparison to WT MsbA, it was determined that these mutants cause reduced cell growth and ATPase activity (19). The CW EPR motional changes are consistent with only inefficient ATP hydrolysis. DEER spectroscopy was used to measure the distance across the dimer, which indicated that both of the mutations cause the purified protein to be trapped in the closed conformation normally only induced by nucleotide, even in the apo state.;The L511P and D512G point mutations previously identified by Polissi et al. (20) in MsbA were also characterized and, like E506Q and H537A, these mutants were found to have significantly reduced cell viability. The L511P mutation also resulted in decreased ATP hydrolysis activity, but the D512G mutation caused a dramatic increase in ATPase activity over WT MsbA (21). CW EPR data indicated that the introduction of both the L511P and D512G mutations results in an altered apo state, demonstrating a change in protein conformation even before the addition of nucleotide. Minimal spectral changes indicate that ATP hydrolysis does occur, but results in only minor motional changes in these point mutations. Given the reduced cell viability and dramatically increased ATPase activity, the D512G mutant appeared to result in protein dysfunction, in which ATP was hydrolyzed at high efficiency, but lipid flipping did not occur.;A270T, a temperature sensitive mutant previously described by Doerrler et al. (22), was characterized to determine the cause of its temperature sensitivity, as well as the source of its dysfunction. A change in amino acid sidechain size, rather than a change in polarity, was demonstrated to be the cause of temperature sensitivity. In addition to the previously described loss of protein function at 44°C, the A270T mutant displayed increased ATPase activity as compared to WT MsbA suggesting that this mutant allowed ATP hydrolysis to continue without lipid transport occurring. CW and pulsed EPR indicate that, while a very subtle local change in protein packing occurs in the presence of nucleotide at the restrictive temperature, there is not a change in global protein conformation due to the A270C substitution.;In summary, five MsbA mutants affecting function, as well as a set of cysteine mutants, were characterized to provide a better understanding of these mutants' roles in protein function. Biochemical techniques were used, as well as EPR spectroscopy, to understand the effect of nucleotide on conserved motifs throughout the NBD using cysteine reporters and to better understand the effects of dysfunctional mutants in protein function by pairing those cysteine reporters with each dysfunctional mutation. |
