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). Lipopolysaccharide transport involves long-range coupling between cytoplasmic and periplasmic domains of the LptB2FGC extractor (Lundstedt et al. 2020).
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)
OstA homologue of 991 aas
OstA homologue of Leptospira interrogans
OstA of 975 aas
OstA of Brachyspira hyodysenteriae
OstA of 537 aas
OstA of Halobacteroides halobius
Putative LptF-LptG-LptD (OstA) fusion protein of 1040 aas (may be an artifact due to a sequencing error, and may also be a contaminant, accounting for its occurance in a Firmicute. However, it shows an N-terminal domain resembling ABC membrane proteins (3.A.1.152) and a hydrophilic C-terminal domain resembling members of porin family 1.B.42. NCBI BLAST results show that there are several homologues of the same "fused" protein in several species of Halothermothrix, Halanaerobium and Candidatus Frackibacter.
OstA of Halothermothrix orenii
LPS export porin complex, LptBCFG-A-DE, consists of LptD (Omp; OmpA; 784 aas)-LptE (RlpB; 193 aas; O.M. lipoprotein)-LptA (KdsD; YhbN; OstA small; 185 aas periplasmic chaparone protein)-LptB (KdsC; YhbG; 241 aas cytoplasmic ABC-type ATPase)-LptC (YrbK, 199aas;1 N-terminal TMS)- LptFG, part of the ABC transporter. LptDE (1:1 stoichiometry) comprise a two-protein β-barrel-lipoprotein complex in the outer membrane that assembles and exports LPS (Chng et al., 2010). After LPS (or a precursor) is transported across the inner membrane by MsbA (3.A.1.106.1), this seven component system translocates LPS from the outer surface of the inner membrane to the outer surface of the outer membrane using ATP hydrolysis to sequentially energize transfer from one binding site to another in several steps (Freinkman et al. 2012; Okuda et al. 2012; Sherman et al. 2014). LPS interacts with LptC and LptA sequentially before being passed to the LptD outer membrane porin, anchored by the LptE lipoprotein on the inner surface of the outer membrane. LptF and LptG are the transmembrane consituents of the ABC pump, and LptB is the ATPase of an ABC-like system that energizes the transport using several ATP molecules (Okuda et al. 2012; Sherman et al. 2014). LptC interconnects the LptBFG ABC system with the periplasmic LptA protein via its large periplasmic domain (Villa et al. 2013). LptDE form a complex in the outer membrane which inserts LPS into this membrane. The 3-D strcture of the complex shows that the LptE lipoprotein inserts into the 26 stranded barrel of LptD as a plug. The first two strands of LptD contain prolines and are therefore distorted, possibly creating a portal for lateral diffusion of LPS into the outer leaflet of the outer membrane (Qiao et al. 2014). The 3-d structure of the Pseudomonas aeruginosa LptA, LptH, has been solved at 2:75 Å resolution revealing a β-jellyroll fold similar to that in LptD (Bollati et al. 2015). Direct interaction of LptB and LptC has been demonstrated (Martorana et al. 2016). A specific binding site in the LptB ATPase for the coupling helices of the transmembrane LptFG complex is responsible for coupling ATP hydrolysis by LptB with LptFG function to achieve LPS extraction (Simpson et al. 2016). After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ABC transporter LptB2FG extracts LPSs from the IM and transports them to the outer membrane. Luo et al. 2017 reported the crystal structure of nucleotide-free LptB2FG from P. aeruginosa. It shows that LPS transport proteins LptF and LptG each contain a TM domain (TMD), a periplasmic beta-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggested that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the beta-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM (Luo et al. 2017). 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 ABC transporter, LptB2FG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane.Li et al. 2019 characterized the structures of LptB2FG and LptB2FGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptB2FGC. LptC inserts its TMS between the two transmembrane domains of LptB2FG, which represents a previously unknown regulatory mechanism for ABC transporters. These results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptB2FG in the inner membrane and Lpt protein interactions in the periplasm (Li et al. 2019). cryo-EM structures of LptB2FG alone and complexed with LptC are known, revealing conformational changes between these states. Two functional transmembrane arginine-containing loops interact with bound AMP-PNP which induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge (Tang et al. 2019). The ABC transporter, LptB2FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge. Lundstedt et al. 2020 showed that residue E86 of LptB is essential for coupling the function of this ATPase to that of its membrane partners, LptFG, at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge consisting of LptC, A and D (from inside to out) and then to the outer membrane insertase, LptE. Defects caused by changing residue E86 are suppressed by mutations altering either the LPS structure or TMSs in LptG. These suppressors fix defects in the coupling helix of LptF, but not of LptG. These observations support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF's coupling helix being important in coordinating cavity collapse with LptB dimerization (Lundstedt et al. 2020).
LptA-G of E. coli:
LptA (YhbN; OstA(s)) (P0ADV1)
LptB (YhbG; ATPase) (P0A9V1)
LptC (YrbG) (P0ADW0)
LptD (OstA; Imp) (P31554)
LptE (RlpB) (P0ADC1)
LptF (YjgP) (P0AF98)
LptG (YjgQ) (P0ADC6)
OstA homologue (Bhat et al. 2011).
OstA homologue of Myxococcus xanthus
OstA of 842 aas
OstA of Rhodopseudomonas palustris
OstA of 753 aas
OstA of Helicobacter pylori
OstA 0f 680 aas
OstA of Hydrogenobaculum sp.
OstA of 880 aas
OstA of Chlorobium luteolum
OstA of 894 aas
OstA of Nonlabens dokdonensis
OstA of 833 aas
OstA of Methylacidiphilum infernorum
OstA homologue of 1069 aa
OstA of Treponema denticola