The Periplasmic Chaperone Skp Facilitates Targeting, Insertion, and Folding of OmpA into Lipid Membranes with a Negative Membrane Surface Potential

Abstract

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.

Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA

“…Although the presence of PG relieved some of the inhibitory effect of PE lipids on OmpA folding, the PE+PG combination had an additive inhibitory effect on OmpLA folding. Our results therefore disagree with previous suggestions that folding of bacterial OMPs is overall favorable in phospholipids with glycerol head groups (25). Unlike the general trend of PE lipids slowing OMP folding, we demonstrated that PG lipids can either kinetically promote or inhibit folding of individual OMPs in the presence of PE.…”

Sequential and spatially restricted interactions of assembly factors with an autotransporter β domain

“…Furthermore, because Skp forms a trimer whose binding cavity appears to be too small to accommodate the entire β domain (37), multiple Skp trimers may interact with the ∼30-kDa fragment. Based on recent in vitro experiments, it is possible that the highly basic Skp protein targets the β domain directly to the negatively charged inner leaflet of the OM (38). However, it also is conceivable that other chaperones that have been implicated in OMP biogenesis (SurA and DegP) bind to the EspP β domain but that these interactions are too transient to be detected by our methods.…”

Sequential Steps in the Assembly of the Multimeric Outer Membrane Secretin PulD

“…A second unresolved question is raised by the discrepancy between the requirement for highly fluid membranes for the folding of outer membrane proteins in vitro (56,57) and the presumed low fluidity of the outer membrane (58). Recent observations indicate that at least certain chaperones can help overcome this barrier (59,60). PulS might play such a role.…”