formation of lipid microdomains, in which PD-BamA accumulated most of the PG lipid present in the membrane and other lipid microdomains containing mostly the zwitterionic lipids PC or PE. It is therefore proposed that binding of PD-BamA leads to lipid phase separations in mixed lipid bilayers.
PD-BamA bound to OmpA at a stoichiometry of 1:1. PD-BamA also bound to BamD, which is the essential lipoprotein of the BAM complex, but at a stoichiometry of two BamD to one PD-BamA. Complexes of PD-BamA with OmpA or with BamD exhibited binding affinities in the nanomolar range of 0.36 nM and 0.34 nM, respectively.
PD-BamA strongly facilitated insertion and folding of OmpA into lipid membranes. Kinetics of PD-BamA mediated folding of OmpA was well described by two parallel folding processes, a fast folding process and a slow folding process, differing by 2-3 orders of magnitude in their rate constants. The folding yields of OmpA depended on the concentration of lipid membranes and also on the lipid head groups. The presence of PD-BamA resulted in increased folding yields of OmpA in negatively charged DOPG, but PD-BamA did not affect the folding kinetics of OmpA into bilayers of zwitterionic but overall neutral lipids. The efficiency of folding and insertion of OmpA into lipid bilayers strongly depended on the ratio PD-BamA/OmpA and was optimal at equimolar concentrations of PD-BamA and OmpA.
The activation energy of the faster folding process of OmpA into charged DOPC/DOPE/DOPG (5:3:2) membranes was determined in the absence and in the presence of PD-BamA to 128 ± 14 kJ/mol and 110 ± 14 kJ/mol, respectively. These activation energies are similar and do not explain the overall faster rates observed in the presence of BamA. Instead, these faster rates resulted from an increased contribution of the faster of the two parallel folding processes when PD-BamA was bound to the membrane surface. The kinetic data suggested a direct interaction between PD-BamA and unfolded OmpA.
To examine complexes of unfolded OmpA with PD-BamA in more detail, site-directed spectroscopy was used to explore contact regions in both, PD-BamA and OmpA. Similar contact regions were then also investigated for another protein complex formed by PD-BamA and the lipoprotein BamD. Mutants of PD-BamA were designed and labeled for fluorescence resonance energy transfer (FRET) experiments to obtain structural information on the protein complexes. A range of single tryptophan and single cysteine PD-BamA mutants were prepared. While tryptophan
itself is a fluorophore, the cysteine is reactive at the sulfhydryl group and can be labeled with a fluorescence probe. To examine proximities for different regions of the binding partners, the donor-acceptor pairs tryptophan-IAEDANS and IAEDANS-5-IAF were used in a range of different point mutants of PD-BamA, OmpA and BamD.
The donor-acceptor pair tryptophan-IAEDANS did not indicate any proximity between PD-BamA mutants and mutants of either OmpA or BamD, as energy transfer was not observed. The Förster distance R0 of this pair of donor and acceptor (~22 Å) was too short to obtain the distances r by which the donors and acceptors were separated in the protein complexes. In contrast, the donor-acceptor pair IAEDANS-5-IAF with a Förster distance of ~46-56 Å showed FRET for various locations of donors and acceptors in PD-BamA and OmpA. Distances that were obtained for the labeled mutants of POTRA domain 3 of PD-BamA and OmpA ranged from 65.4 Å to 131.2 Å. The smallest separations were observed for the mutation L223C in PD-BamA, which is located at the top of the groove in POTRA domain 3 at the edge next to POTRA domain 4.
The distances that were obtained by intermolecular FRET studies for the FRET pairs in mutants of POTRA domain 5 of PD-BamA and OmpA were in the range from 48.9 Å to 106.0 Å. The shortest distances were observed for the mutation N390C in PD-BamA, which is located close to the BamA β-barrel next to transmembrane β-strands β1 and β16. The distances for FRET donor-acceptor pairs of POTRA domain 5 of PD-BamA and BamD were found to be between 73.4 Å to 162.9 Å. Highest FRET was observed for mutant A363C-PD-BamA, with the mutation located on the α-helix α1 and BamD mutant Q230C, with the mutation located on the C-terminal α-helix of TPR segment 5. These data suggest, that the site of interaction on PD-BamA for OmpA might be oriented towards the exterior environment away from the preceding POTRA domains, but that PD-BamA is oriented with its short α-helix α1 of POTRA domain 5 towards the C-terminal end of BamD.
Changes in fluorescence intensities upon binding of single tryptophan mutants of PD-BamA to lipid membranes (DOPC/DOPE/DOPG (5:3:2)) could be observed, indicating that the presence of lipid membranes causes conformational changes in the structure of PD-BamA. Strongest changes in the fluorescence intensities were measured for mutations located in the region of POTRA domain 5, that comprises the
PD-BamA interacts and binds to the substrate protein OmpA most probably via its POTRA domain 5, it plays an important role in membrane dynamics and facilitates folding and insertion of OmpA into negatively charged membranes. Thus, the function of the periplasmic domain of BamA in the BAM complex improves the understanding of the fundamental principles of the membrane assembly of β-barrel outer membrane proteins.