We have utilized mPEG-mal labeling in these studies to demonstrate that ArnT derived from S. typhimurium does not contain disulfide bonds and that all eight native cysteines are freely reactive. This was somewhat surprising given the large number of cysteines present, however the cytoplasm is a reducing environment, so it would not be unexpected that any cytoplasmic cysteines would be reduced. In addition, the putative topology model of ArnT suggests that several cysteines may be located within transmembrane helical segments (), and it is unusual for disulfides to form between membrane helices. We also observed that the majority of the protein fraction was labeled with six, seven or eight mPEG-mal moieties. In addition, none of the protein was left unmodified, indicating reasonable accessibility of each of the cysteine locations to this fairly large modifying reagent, even to the presumably buried, hydrophobic helical sites.
The absence of disulfide bonds in ArnT is further supported by the fact that the protein tolerated the substitution of all eight native cysteine residues (i.e., 2A6S) and produced a folded and functional protein. Although the 3-D structure of ArnT is not yet known, our CD and growth assay data show that none of the native cysteines are required for structural integrity or in vivo
complementation. While the removal of all eight cysteines (2A6S) or the partial substitution of the native cysteines (2C6S) generated proteins able to complement the chromosomal knockout, the substitution of all eight cysteines with serines (8S) resulted its inability to sustain cell growth in the presence of polymyxin. Serine is typically a suitable isosteric substitute for cysteine in protein mutagenesis, yet this change at C148 and C149 in the 6S ArnT makes the entire protein inactive in vivo
, while alanine mutations at these sites (2A6S) retain sufficient activity to complement the chromosomal knockout of ArnT. This suggests that proper folding of ArnT may require a hydrophobic residue such as alanine or cysteine at these positions (23
), yet the partially native protein 4S4C remained functional in vivo
with serines at the C148/C149 positions. Our previous experience with visual rod arrestin also required the use of a combination of alanine and serine substitutions to obtain an intact cysteine-free protein (24
Next we identified a large stretch of amino acids between 234 and 256 that are required for proper function of ArnT, as assumed through complementation of the chromosomal knockout of ArnT as assessed in the in vivo growth assay, indicating that the proposed loop comprised of sites 229-256 is likely critical to either lipid A or the undecaprenyl phosphate-L-Ara4N recognition and binding and/or to the transfer of the L-Ara4N onto the lipid A moiety. The topology model suggests that these sites form an interhelical loop, thus we speculate that this region may be involved in recognizing and/or binding one or both of the lipid substrates after being flipped to the periplasmic leaflet of the inner membrane. This region contains nearly half polar or charged amino acids, which would be consistent with sites in this region being involved in the recognition of the lipid A phosphate groups and transfer of L-Ara4N. Of those mutants that retained the ability to confer resistance to polymyxin, two were particularly interesting; the W240C and K255C proteins were expressed at very low levels of protein within the membrane. It is possible that the mutation at these sites affects the efficiency of protein folding and/or membrane insertion, which lowers the amount of protein present in the membrane fraction, yet once integrated into the membrane the protein is still able to complement the knockout. Conversely, two other mutations (241 and 242) had low, but non-zero, levels of protein expression (9-13%) yet showed negative growth assay results. It is possible that these mutations also affect the efficiency of protein folding and/or membrane insertion, lowering the amount of protein present in the membrane fraction, but that once integrated still negatively affect protein function by interfering with or preventing substrate binding or transfer.
The above examples of small amounts of protein present in the membrane fraction highlight the fact that very low levels of functional ArnT are necessary for cellular protection against polymyxin-induced killing. It is remarkable that even at <0.1% of the total membrane protein, functional ArnT proteins can modify sufficient lipid A to complement the chromosomal knockout by completely protecting the cell from the levels of polymyxin used in our assay.
In our survey of 31 consecutive sites in this protein we have discovered a number of critical side chains within ArnT particularly clustered between residues 234 and 256. Our results indicating that the ability of sites 257-260, all of which are aromatic in the native protein, to complement the chromosomal knockout in the growth assay upon substitution to cysteine is consistent with a model in which these sites are located within a membrane-spanning helix and not within a functionally critical soluble loop. It is tempting to relocate sites 230-233 on the opposite end of the region studied for similar reasons, but three of the four are highly polar and thus are not likely located within a hydrophobic membrane helix.
In summary, we have established here that all eight of the native cysteines in S. typhimurium ArnT are in the reduced form and accessible to labeling by mPEG-mal and have created a cysteine-free protein that is both structurally and functionally intact. While alanine substitution has proven useful in the early stages of protein function research, cysteine substitutions provide the opportunity for attachment of a molecular probe in future studies. Using this approach, we have identified for the first time 14 critical residues within a consecutive 31-residue span that are essential for function of ArnT and 3 additional residues that completely disrupt protein folding or insertion into the bacterial inner membrane. Our ability to generate a cysteine-free construct of ArnT allowed us to conduct cysteine-scanning mutagenesis analysis of protein expression and function and sets the stage for additional studies of the structure and function of this protein using biochemical and biophysical approaches based on site-directed cysteine labeling.