Outer membrane protein A (OmpA)

1.4.1.1 Structure

OmpA is an abundant structural protein of the outer membrane of Escherichia.coli and occurs at about 100 000 copies/cell [Sonntag et al. 1978]. This 325-residue, heat-modifiable protein contains two domains: transmembrane (TM) domain and periplasmic domain. It is believed to connect the outer membrane structurally to the periplasmic peptidoglycan layer via its globular periplasmic domain, which consists of residues ~172-325.

Residues 1-171 form the transmembrane domain, whose structure has been solved by X-ray crystallography [Pautsch et al. 1998] and NMR [Arora et al. 2001a]. It forms an

all-next-Interaction of OMPs with Skp and LPS

neighbor antiparallel eight-stranded β-barrel (Figure 1.8). All strands are tilted by ~45°

relative to the membrane normal. Four long and mobile extracellular loops and three short periplasmic chain turns correspond well with the general porin structure [Schulz 1996].

1.4.1.2 Functions

The basic function of OmpA appears to hold peptidoglycan and the outer membrane together as a whole structure in E. coli [Koebnik 1995]. Clearly some phages [Morona et al.

1985] and colicin [Foulds et al. 1978] are able to use OmpA for docking. OmpA also happens to interact with brain microvascular endothelial cells (BMEC) that promotes E. coli invasion of BMEC [Prasadarao et al. 1996]. Some small molecules may pass the β-barrel of OmpA and cross the outer membrane [Arora et al. 2000].

1.4.1.3 Refolding

A decisive step in the biosynthesis of many secretory and plasma membrane proteins is their transport across the endoplasmic reticulum (ER) membrane in eukaryotes or across the cytoplasmic membrane in prokaryotes. In co-translational translocation, the major partner is the ribosome. The elongating polypeptide chain moves directly from the ribosome into the associated membrane channel formed by the Sec61p complex in eukaryotes and SecY complex in eubacteria and archaea. In post-translational translocation, polypeptides are biosynthesized in the cytosol and then transported across the membrane. How membrane proteins insert and fold into the outer membrane of bacteria after translocation is largely unknown.

In vitro, both classes of integral membrane proteins (IMPs) require either detergent micelles or lipid bilayers for folding. The folding of IMPs into detergent micelles was mostly studied with bacteriorhodopsin (BR) of Halobacterium salinarium [Engelman et al. 1981;

Huang et al. 1981; Popot et al. 1987; Kahn et al. 1992; Booth et al. 1999], OmpA [Dornmair et al. 1990; Kleinschmidt et al. 1999a], OmpF [Surrey et al. 1996], OmpG [Conlan et al.

2003], PhoE [de Cock et al. 1996] and AIDA [Mogensen et al. 2005a]. Schweizer et al.

[Schweizer et al. 1978] showed for the first time that the 8-stranded β-barrel OmpA partially regained native structure in presence of lipopolysaccharide and Triton-X-100 after dilution of the denaturants SDS or urea. Similarly, Dornmair et al. [Dornmair et al. 1990] demonstrated that after heat-denaturation in sodium dodecyl sulfate (SDS) micelles, OmpA can refold into micelles of the detergent octylglucoside in absence of LPS. Surrey and Jähnig [Surrey et al.

1992] showed first that OmpA spontaneously inserts and folds into phospholipid bilayers.

Interaction of OMPs with Skp and LPS

Completely unfolded and solubilized OmpA in 8 M urea was refolded upon strong dilution of the denaturant in presence of small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (diC14PC). These studies suggest that the information for the formation of native structure in integral membrane proteins is contained in their amino acid sequence, as previously described by the Anfinsen paradigm for soluble proteins [Anfinsen 1973], but requires the hydrophobic environment of micelles or bilayers.

The concerted mechanism of OmpA insertion into bilayers was first demonstrated by time-resolved Trp fluorescence quenching (TDFQ) [Kleinschmidt et al. 1999c]. A tentative model of OmpA folding is shown in Figure 1.9.

Figure 1.9 Folding model of OmpA

The kinetics of β-sheet secondary and β-barrel tertiary structure formation in OmpA have the same rate constants and are coupled to the insertion of OmpA into the lipid bilayer. The locations of the five tryptophans in the three identified membrane-bound folding intermediates and in the completely refolded state of OmpA are shown. Additional details, such as the translocation of the long polar loops across the lipid bilayers must still be determined. The figure was adapted from [Kleinschmidt 2003].

In recent years, periplasmic chaperones were identified that play an important role in the assembly steps of outer membrane proteins in vivo or in vitro. Direct biochemical evidence for a chaperone-assisted three-step delivery pathway of OmpA to a model membrane was first given by Bulieris et al [Bulieris et al. 2003]. It was demonstrated that the

Interaction of OMPs with Skp and LPS

periplasmic chaperone Skp keeps OmpA soluble in vitro at pH 7 in an unfolded form even when the denaturant urea was diluted out. Skp was also shown to prevent the premature folding of OmpA into LPS that is also present in the periplasm and to inhibit the folding of OmpA into phospholipid bilayers. Only when Skp complexes with unfolded OmpA were reacted with LPS in a second stage, a folding competent form of OmpA was formed that efficiently inserted and folded into phospholipid bilayers in a third stage. The interaction of the OmpA/Skp/LPS complex with the lipid bilayer is apparently the most important event to initiate folding of OmpA in presence of chaperones and LPS as folding catalysts. The described assisted folding pathway and discovered 3:1 stoichiometry for Skp binding to OmpA was later supported by the observation that Skp is trimeric in solution [Schlapschy et al. 2004] and by the description of the crystal structure of Skp and a putative LPS binding site in Skp. One LPS binding site per Skp monomer is consistent with the observation of optimal folding kinetics of OmpA from an OmpA/Skp/LPS complex at 0.5–1.7 mol LPS mol Skp^–1 [Bulieris et al. 2003]. In this case, a 1: 1 stoichiometry perhaps indicates that LPS only binds to the LPS binding site of Skp and OmpA is completely shielded from interactions with LPS.

A current folding model for this assisted OmpA folding pathway is shown in Figure 1.10.

Figure 1.10 Scheme for an assisted folding pathway of a bacterial outer membrane protein

OmpA is translocated through the cytoplasma membrane in an unfolded form (U) and binds to a small number of molecules of the periplasmic chaperone Skp, which solubilizes OmpA in the unfolded state (USkp3). The complex of unfolded OmpA and Skp interacts with a small number of LPS molecules to form a folding competent intermediate of OmpA (FCSkp3LPSn). In the final step, folding competent OmpA inserts and folds into the lipid bilayer [Kleinschmidt 2003].

Interaction of OMPs with Skp and LPS