1.B.42 The Outer Membrane Lipopolysaccharide Export Porin (LPS-EP) Family
Lipopolysaccharides (LPSs) coat the outer surfaces of the outer membranes of Gram-negative bacteria. LPS is synthesized in the bacterial inner membrane. The lipid A-core region and the O-antigen side chain are synthesized and transported separately to the outer periplasmic surface of the inner membrane where the O-antigen chain is ligated to the lipid A core. The fully assembled LPS is then transported to the outer surface of the outer membrane. The proteins that appears to catalyze this transport process includes a large, essential, outer membrane protein which when defective results in formation of aberrant membranes. It has been called 'increased membrane permeability' (Imp) or 'organic solvent tolerance protein' (OstA). Mutants defective for this protein in Neisseria meningitidis are viable but produce reduced amounts of LPS. That which is produced is restricted to the periplasmic space. This protein is highly conserved among Gram-negative bacteria.
The LptB2FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. Simpson et al. 2016 identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport. Transport involves a stable association between the inner (LptBFG) and outer (LptDE) membrane components, supporting a mechanism in which lipopolysaccharide molecules are pushed one after the other across a protein bridge (LptCA) that connects the inner and outer membranes (Sherman et al. 2018).
The Lpt system consists of seven known LPS transport proteins (LptA-G) spanning from the cytoplasm to the cell surface. Imp (OstA or LptD) of E. coli forms a complex with another essential protein, RlpB (LptE) (Takase et al., 1987). LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. LptE binds LPS specifically and may serve as a substrate recognition site at the OM (Chng et al., 2010). RlpB is a rare lipoprotein that is essential for viability and for transport of LPS to the outer surface of the outer membrane (Wu et al., 2006). The seven proteins required for LPS export have been reported to form a transenvelope complex spanning the peptidoglycan layer as well as the two membranes of the Gram-negative envelope (Chng et al., 2010b). The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel, where LptD is the outer membrane barrel, and LptE is the plug (Freinkman et al., 2011). The periplasmic component, LptA is able to form a stable complex with the inner membrane anchored LptC but does not interact with the outer membrane anchored LptE (Bowyer et al., 2011). The LptC component of the LptBFGC complex may act as a dock for LptA, allowing it to bind LPS after it has been assembled at the inner membrane. That no interaction between LptA and LptE has been observed supports the theory that LptA binds LptD in the LptDE homodimeric complex at the outer membrane.
LptD shows cation selectivity and has an estimated pore diameter of 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which suggests that LptD is able to bind and transport lipid A. LptD proteins of different bacteria all have periplasmic N-terminal domains and C-terminal barrel regions. The latter show distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. LptD from Anabaena sp. PCC 7120 can also transport Lipid A (Haarmann et al., 2010).
To date, the only proteins implicated in LPS transport are MsbA (TC# 3.A.1.106), responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Two additional Escherichia coli essential genes, yhbN and yhbG, renamed lptA and lptB, respectively, participate in LPS biogenesis (Sperandeo et al., 2007). Mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. LptA is located in the periplasm, and expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli, possibly together with Imp/RlpB. A unique LptA structure reported by Suits et al (2008) represents a novel fold, consisting of 16 consecutive antiparallel beta-strands, folded to resemble a slightly twisted β-jellyroll. Each LptA molecule interacts with an adjacent LptA molecule in a head-to-tail fashion to resemble long fibers.
OstA (Imp) homologues have been shown to play a role in outer membrane biogenesis. Bioinformatic analyses of these proteins in organisms with fully sequenced genomes reveal that these proteins occur only in bacteria with two membranes. Two OstA types were identified, large OstAs (L; 812 ± 94 residues) and small OstAs (S; 181 ± 25 residues) (Hu and Saier, 2006). S possesses only the OstA domain while L has this domain plus a larger nonhomologous OstA-C domain. Bacteria lacking both S and L proteins were primarily restricted to reduced genome size pathogens and symbionts. Several of these bacteria appear to also have incomplete sets of genes required for the biosynthesis of typical Gram-negative bacterial lipopolysaccharide (LPS). Phylogenetic analyses of both S and L homologues showed that they generally follow the phylogenies of the 16S rRNAs from the same organisms with few exceptions. They may comprise two orthologous sets of proteins that together facilitate a single unified function. While most organisms possess a single L and a single S, those lacking S but possessing L are more numerous than those lacking L but possessing S. Based on these findings, is was suggested that the L and S proteins normally act together in macromolecular insertion, (1) they are important for proper LPS assembly in the outer leaflet of the outer membrane, (2) they function specifically to export LPS to the outer leaflet, and (3) L provides a primary function while S provides an important auxiliary function (Hu and Saier, 2006).
There are at least 5 proteins that may make up the machinery for transport from the periplasmic surface of the inner membrane to the outer surface of the outer membrane. The four that have been known for some time were called (1) OstA(L) or Imp (Hu and Saier, 2006) (an outer membrane protein), (2) RlpB, a LPS-assembly lipoprotein discussed above, (3) OstA(S) or LptA (a periplasmic protein showing homology to part of OstA(L)), and (4) LptB, a presumed cytoplasmic ATPase, homologous to ATPases of the ABC superfamily (Hu and Saier, 2006; Sperandeo et al., 2006). Sperandeo et al. (2008) have identified a fifth component, and have provided a uniform nomenclature of the complex. While the inner membrane (IM) transport protein MsbA, is responsible for flipping LPS across the IM, five components of the LPS transport machinery downstream of MsbA have been identified. These are: The OM protein complex LptD/LptE (formerly Imp/RlpB, respectively), the periplasmic LptA protein, the IM associated cytoplasmic ABC protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery. Depletion of any of the above proteins leads to common phenotypes i) appearance of abnormal membrane structures in the periplasm; ii) accumulation of de novo synthesized LPS in two membrane fractions with lower density than the OM; iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of IM. Thus, LptA, LptB, LptC, LptD and LptE operate in the LPS assembly pathway (Sperandeo et al., 2008). The LptA protein of Escherichia coli is a periplasmic lipid A binding protein involved in the lipopolysaccharide export pathway (Tran et al., 2008).
E. coli contains two proteins, previously of unknown function called YjgP and YjgQ. They are about 350aas long and have 6TMSs in a 3+3 arrangement, where the two 3TMS units are separated by a large hydrophilic domain (150 residues, including an OstA domain). They are found mainly in Gram-negative bacteria and are annotated as 'putative permease protein'. The YjgPQ proteins have over a thousand sequenced homologues present in prokaryotes. The first transmembrane domain is COG0795. Although YjgP and YjgQ have the same topology, they are substantially sequence divergent.
The two essential Escherichia coli IM proteins, YjgP and YjgQ, are required for the transport of LPS to the cell surface (Ruiz et al., 2008). These two proteins, have been renamed LptF and LptG, respectively. They are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM (Ruiz et al., 2008). However Narita and Tokuda (2009) have concluded that LptBFGC comprise an ABC transpoter (3.A.1) that is required in outer membrance lipopolysaccharide sorting. While LptB is homologous to the ATP hydrolyzing subunits of ABC transporters, LptF (COG0795), LptG (COG0795) and LptC (DUF1239) are not demonstrably homologous to ABC type subunits. This raises some doubt but does not negate the evidence.
LptA-G (1.B.42.1.2) form a bridge between the inner and outer membranes of gram-negative bacteria. Freinkman et al. (2012) used in vivo photo-cross-linking to reveal the specific protein-protein interaction sites that give rise to the Lpt bridge and also showed that the formation of this transenvelope bridge cannot proceed before the correct assembly of the LPS translocon in the OM. This ordered sequence of events may ensure that LPS is never transported to the OM if it cannot be translocated across it to the cell surface.
LptD translocates LPS from the periplasm across the outer membrane (OM). In E. coli, this protein contains two disulfide bonds and forms the OM LPS translocon with the lipoprotein LptE. Chng et al. (2012) identified seven in vivo states on the oxidative folding pathway of LptD. Proper assembly involved a nonfunctional intermediate containing nonnative disulfides. Intermediate formation required the oxidase DsbA, and subsequent maturation to the active form with native disulfides was triggered by LptE. Thus, disulfide bond-dependent protein folding of LptD requires the proper assembly of a two-protein complex in order to promote disulfide bond rearrangement (Chng et al., 2012).
The transport reaction catalyzed by the Lpt complex is:
LPS (inner membrane) LPS (outer membrane)