The basic biochemical and biophysical principles by which chaperone-bound membrane proteins are targeted to the outer membrane of Gram-negative bacteria for insertion and folding are unknown. Here we compare spontaneous folding of outer membrane protein A (OmpA) of Escherichia coli from its urea-unfolded form and from the complex with its periplasmic chaperone Skp into lipid bilayers. Skp facilitated folding of OmpA into negatively charged membranes containing dioleoylphosphatidylglycerol (DOPG). In contrast, Skp strongly inhibited folding of OmpA when bilayers were composed of dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine (DOPC). These results indicate that the positively charged Skp targets OmpA to a negatively charged membrane, which facilitates the release of OmpA from its complex with Skp for subsequent folding and membrane insertion. The dual functionality of Skp as a chaperone and as a targeting protein is ideal to mediate the transport of OmpA and other outer membrane proteins across the periplasm in a folding-competent form to the outer membrane, which is negatively charged on its periplasmic side. OmpA (pI 5.5) folded most efficiently above its isoelectric point. In the absence of Skp and in contrast to folding into DOPC bilayers, insertion and folding of OmpA were retarded for membranes containing DOPG at neutral or basic pH because of electrostatic repulsion. When folding of OmpA was performed near its isoelectric point, urea dilution led to a more compact aqueous form of OmpA previously characterized by fluorescence, which folded at a much slower rate. Under conditions where two different aqueous conformations of OmpA coexisted, e.g., in the titration region of OmpA, the last step of OmpA folding could be well described by two parallel pseudo-first-order kinetic phases. In this kinetic model, the contribution of the faster folding process, but not the changes in the rate constants, determined the folding yields obtained at different pH. The faster phase dominated when the experimental conditions favored the less compact form of aqueous OmpA.

BackgroundThe inner membrane-anchored periplasmic folding factor PpiD is described as a parvulin-like peptidyl prolyl isomerase (PPIase) that assists in the maturation of the major beta-barrel outer membrane proteins (OMPs) of Escherichia coli. More recent work however, calls these findings into question. Here, we re-examined the role of PpiD in the E. coli periplasm by analyzing its functional interplay with other folding factors that influence OMP maturation as well as general protein folding in the periplasmic compartment of the cell, such as SurA, Skp, and DegP.ResultsThe analysis of the effects of both deletion and overexpression of ppiD on cell envelope phenotypes revealed that PpiD in contrast to prior observations plays only a minor role, if any, in the maturation of OMPs and cannot compensate for the lack of SurA in the periplasm. On the other hand, our results show that overproduction of PpiD rescues a surA skp double mutant from lethality. In the presence of increased PpiD levels surA skp cells show reduced activities of both the SigmaE-dependent and the Cpx envelope stress responses, and contain increased amounts of folded species of the major OMP OmpA. These effects require the anchoring of PpiD in the inner membrane but are independent of its parvulin-like PPIase domain. Moreover, a PpiD protein lacking the PPIase domain also complements the growth defects of an fkpA ppiD surA triple PPIase mutant and exhibits chaperone activity in vitro. In addition, PpiD appears to collaborate with DegP, as deletion of ppiD confers a temperature-dependent conditional synthetic phenotype in a degP mutant.ConclusionsThis study provides first direct evidence that PpiD functions as a chaperone and contributes to the network of periplasmic chaperone activities without being specifically involved in OMP maturation. Consistent with previous work, our data support a model in which the chaperone function of PpiD is used to aid in the early periplasmic folding of many newly translocated proteins.

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Periplasmic Skp facilitates folding and membrane insertion of many outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. We have examined the binding sites of outer membrane protein A (OmpA) from Escherichia coli in its complexes with the membrane protein chaperone Skp and with Skp and lipopolysaccharide (LPS) by site-directed fluorescence spectroscopy. Single-Trp OmpA mutants, W(n)-OmpA, with tryptophan at position n in the polypeptide chain were isolated in the unfolded form in 8 M urea. In five beta(x)W(n)-OmpA mutants, the tryptophan was located in beta-strand x, in four l(y)W(n)-OmpA mutants, in outer loop y, and in three t(z)W(n)-OmpA mutants in turn z of the beta-barrel transmembrane domain (TMD) of OmpA. PDW(286)-OmpA contained tryptophan in the periplasmic domain (PD). After dilution of the denaturant urea in aqueous solution, spectra indicated a more hydrophobic environment of the tryptophans in beta(x)W(n) mutants in comparison to l(y)W(n)-OmpA and t(z)W(n)-OmpA, indicating that the loops and turns form the surface of hydrophobically collapsed OmpA, while the strand regions are less exposed to water. Addition of Skp increased the fluorescence of all OmpA mutants except PDW(286)-OmpA, demonstrating binding of Skp to the entire beta-barrel domain but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands beta(1) to beta(3) in the N-terminus than with strands beta(5) to beta(7) in the C-terminus. This asymmetry was not observed for the outer loops and the periplasmic turns of the TMD of OmpA. The fluorescence results demonstrated that all turns and loops l(1), l(2), and l(4) were as strongly bound to Skp as the N-terminal beta-strands. Addition of five negatively charged LPS per one preformed Skp.W(n)-OmpA complex released the C-terminal loops l(2), l(3), and l(4) of the TMD of OmpA from the complex, while its periplasmic turn regions remained bound to Skp. Our results demonstrate that interactions of Skp.OmpA complexes with LPS change the conformation of OmpA in the Skp complex for facilitated insertion and folding into membranes.