1.B.46 The Outer Membrane LolAB Lipoprotein Insertion Apparatus (LolAB) Family

LolA is a periplasmic chaperone protein of E. coli that translocates lipoproteins destined for the outer membrane from the inner membrane to the outer membrane protein, LolB, which appears to insert the substrate lipoprotein into the outer membrane. LolB is essential for bacterial growth and lipoprotein insertion (Tanaka et al., 2001). LolA shows limited but significant sequence similarity to the autotransporter (AT) protein of the Helicobacter mustelae surface ring protein, Hsr (TC #1.B.12.7.1). LolB shows slight sequence similarity to a central portion of the H. influenzae protective surface antigen D15 (TC #1.B.33.1.2). The crystal structures of both LolA and LolB have been solved (Takeda et al., 2003). They show similar folds. Homologues of LolA include LppX which is thought to translocate phthiocerol dimycocerosates (DIM) to the outer membranes of mycobacterial species (Sulzenbacher et al., 2006).  Outer membrane lipoproteins and their export has been reviewed (Wilson and Bernstein 2015).

An area in the same regions of LolA and LolB mediate LolA/LolB interaction with each other (Okuda and Tokuda, 2009). This area is located at the entrance of the hydrophobic cavity. This area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of the lipoproteins through Lol proteins occurs in a mouth-to-mouth manner (Okuda and Tokuda, 2009). The LolA-LolB complex appears to form a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected. This enables lipoproteins to be transferred from LolA to LolB. Opening and closing of the hydrophobic cavity of LolA couple to lipoprotein binding and release (Oguchi et al., 2008).

Sorting of E. coli lipoproteins to the outer membrane is catalyzed by the Lol system composed of five proteins, LolA through LolE. An ATP-binding cassette transporter LolCDE complex (3.A.1.125.1; see Watanabe et al., 2007) recognizes a newly synthesized outer membrane-directed lipoprotein on the periplasmic leaflet of the inner membrane and mediates the detachment of lipoproteins from the inner membrane in the presence of a periplasmic carrier protein, LolA. LolA forms a soluble complex with one molecule of lipoprotein and traverses the periplasmic space to reach a lipoprotein-specific outer membrane receptor, LolB. LolB, itself an outer membrane lipoprotein, receives a lipoprotein from LolA and then incorporates it into the outer membrane (Tanaka et al., 2007). A short helix in the C-terminal region of LolA is important for the specific membrane localization of lipoproteins (Okuda et al., 2008).

The role of a substrate lipoprotein Asp residue at position 2 in lipoprotein sorting has been extensively studied. The Asp residue functions as a LolCDE avoidance signal because LolCDE does not recognize lipoproteins with Asp at position 2. Both the negative charge of the Asp residue and the positive charge of phosphatidylethanolamine are critical for the LolCDE avoidance.

E. coli lipoproteins are sorted to the outer membrane by default. Only Asp at position 2 actively functions as an intrinsic inner membrane retention signal. However, lipoproteins of Pseudomonas aeruginosa are sorted according to the residues at positions 3 and 4. The five Lol proteins are responsible for the sorting of lipoproteins to the outer membrane of P. aeruginosa, as in the case of E. coli lipoproteins, but differences in lipoprotein sorting signals reflect differences in the properties of LolCDE in these two organisms (Tanaka et al., 2007).  These transporters may be parts of nanomachines ().

Escherichia coli lipoproteins are localized to either the inner or the outer membrane depending on the residue that is present next to the N-terminal acylated Cys. Asp at position 2 causes the retention of lipoproteins in the inner membrane. In contrast, the residues at positions 3 and 4 determine the membrane specificity of lipoproteins in Pseudomonas aeruginosa. The five Lol proteins involved in the sorting of E. coli lipoproteins are conserved in P. aeruginosa. The LolCDE homologue thus purified has been reconstituted into proteoliposomes with lipoproteins (Tanaka et al., 2007). When incubated in the presence of ATP and a LolA homologue, the reconstituted LolCDE homologue released lipoproteins, leading to the formation of a LolA-lipoprotein complex. Lipoproteins were then incorporated into the outer membrane depending on a LolB homologue. As revealed in vivo, lipoproteins with Lys and Ser at positions 3 and 4, respectively, remained in proteoliposomes. On the other hand, E. coli LolCDE released lipoproteins with this signal and transferred them to LolA of not only E. coli but also P. aeruginosa. Thus, Lol proteins are responsible for the sorting of lipoproteins to the outer membrane of P. aeruginosa, as in the case of E. coli, but they respond differently to inner membrane retention signals.

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. 1.21 A resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB (Ryan et al., 2008). For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE identified the active site and five residues important for binding or catalysis. VioE may act as a catalytic chaperone, using a fold previously associated with lipoprotein transporters, to catalyze the production of its prodeoxyviolacein product (Ryan et al., 2008).

The reaction believed to be catalyzed by the LolAB pair is:

lipoprotein (inner membrane) → lipoprotein (outer membrane).