Concentration-dependent oligomerization and oligomeric arrangement of LptA.

Gram-negative bacteria such as Escherichia coli have an inner membrane and an asymmetric outer membrane (OM) that together protect the cytoplasm and act as a highly selective permeability barrier. Lipopolysaccharide (LPS) is the major component of the outer leaflet of the OM and is essential for the survival of nearly all Gram-negative bacteria. Recent advances in understanding the proteins involved in the transport of LPS across the periplasm and into the outer leaflet of the OM include the identification of seven proteins suggested to comprise the LPS transport (Lpt) system. Crystal structures of the periplasmic Lpt protein LptA have recently been reported and show that LptA forms oligomers in either an end-to-end arrangement or a side-by-side dimer. It is not known if LptA oligomers bridge the periplasm to form a large, connected protein complex or if monomeric LptA acts as a periplasmic shuttle to transport LPS across the periplasm. Therefore, the studies presented here focus specifically on the LptA protein and its oligomeric arrangement and concentration dependence in solution using experimental data from several biophysical approaches, including laser light scattering, crosslinking, and double electron electron resonance spectroscopy. The results of these complementary techniques clearly show that LptA readily associates into stable, end-to-end, rod-shaped oligomers even at relatively low local protein concentrations and that LptA forms a continuous array of higher order oligomeric end-to-end structures as a function of increasing protein concentration.

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

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.

Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA.

MsbA is a member of the ABC transporter superfamily and is homologous to ABC transporters linked to multidrug resistance. The nucleotide binding domains (NBDs) of these proteins include conserved motifs that are involved in ATP binding, including conserved SALD residues (D-loop) that are diagnostic in identifying ABC transporters but whose roles have not been identified. Within the D-loop, single point mutations L511P and D512G were discovered by random mutational analysis of MsbA to disrupt protein function in the cell [Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233] but have not been further studied in MsbA or in detail in any other ABC transporter. In these studies, we show that both L511P and D512G mutants of MsbA are able to bind ATP at near-wild-type levels but are unable to maintain cell viability in an in vivo growth assay, verifying the theory that they are dysfunctional at some point after ATP binding. An ATPase assay further suggests that the L511P mutation prevents effective ATP hydrolysis, and an ATP detection assay reveals that only small amounts of ATP are hydrolyzed; D512G is able to hydrolyze ATP at a rate 3-fold faster than that of the wild type. EPR spectroscopy studies using reporter sites within the NBDs also indicate that at least some hydrolysis occurs in L511P or D512G MsbA but show fewer spectral changes than observed for the same reporters in the wild-type background. These studies indicate that L511 is necessary for efficient ATP hydrolysis and D512 is essential for conformational rearrangements required for flipping lipid A.

Identification of a functionally important loop in Salmonella typhimurium ArnT.

ArnT confers resistance to the antibiotic polymyxin in Salmonella typhimurium and Escherichia coli through the modification of lipid A, a major component of the outer surface of gram-negative bacteria. ArnT transfers a neutral aminoarabinose moiety onto the negative phosphate groups of lipid A, reducing the surface charge of the bacteria and preventing cationic peptides such as polymyxin from electrostatically recognizing and killing the bacteria. We previously reported the first expression, purification, and functional analysis of ArnT from S. typhimurium [Bretscher, L. E., Morrell, M. T., Funk, A. L., and Klug, C. S. (2006) Protein Expression Purif. 46, 33-39]. Our studies showed that ArnT is highly alpha-helical and described a new in vivo functional growth assay. Here, we use the cysteine-specific mPEG-mal to demonstrate that all eight of the native cysteines in S. typhimurium ArnT are in the reduced form and not involved in disulfide bonds and show that the cysteine-free protein is structurally and functionally intact as characterized by circular dichroism and the in vivo growth assay. Following this initial characterization, in vivo expression and function profiles were surveyed for 31 consecutive mutations within a putative ArnT loop. These studies identify for the first time 14 residues that are essential for function of the ArnT transferase and 3 additional residues that completely disrupt protein folding or insertion into the bacterial inner membrane.

Functionally important ATP binding and hydrolysis sites in Escherichia coli MsbA.

ATP-binding cassette (ABC) transporters make up one of the largest classes of proteins found in nature, and their ability to move a variety of substrates across the membrane using energy from the binding or hydrolysis of ATP is essential to an array of human pathologies and to bacterial viability. MsbA is an essential ABC transporter that specifically transports lipid A across the inner membranes of Gram-negative organisms such as Escherichia coli. The exact mechanisms of function during the binding and hydrolysis of ATP at the molecular level remain unclear. The studies presented and summarized in this work directly address the role and local dynamics of specific residues within the conserved ABC motifs in E. coli MsbA using in vivo growth and biochemical activity assays coupled with site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy motional and accessibility analysis. This first comprehensive analysis of the specific residues in these motifs within MsbA indicates that closure of the dimer interface does not occur upon ATP binding in this transporter.