In Gram-negative bacteria, the assembly of β-barrel outer-membrane proteins (OMPs) requires the β-barrel-assembly machinery (BAM) complex. We determined the crystal structure of the 200-kDa BAM complex from Escherichia coli at 3.55-Å resolution. The structure revealed that the BAM complex assembles into a hat-like shape, in which the BamA β-barrel domain forms the hat's crown embedded in the outer membrane, and its five polypeptide transport-associated (POTRA) domains interact with the four lipoproteins BamB, BamC, BamD and BamE, thus forming the hat's brim in the periplasm. The assembly of the BAM complex creates a ring-like apparatus beneath the BamA β-barrel in the periplasm and a potential substrate-exit pore located at the outer membrane-periplasm interface. The complex structure suggests that the chaperone-bound OMP substrates may feed into the chamber of the ring-like apparatus and insert into the outer membrane via the potential substrate-exit pore in an energy-independent manner.

The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3 ecto ), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2 Fu1-Fu2 ), and Rspo2 Fu1-Fu2 in complex with ZNRF3 ecto , or RNF43 ecto . A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3 ecto and RNF43 ecto surface. Rspo binding enhances dimerization of ZNRF3 ecto but not of RNF43 ecto .Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2.

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 Å resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the β barrel is composed of 5 markedly short β strands, and the loops stemming from these β strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA β barrel and structure-based mutagenesis studies suggest that the transmembrane β strands of OMP substrates may integrate into the outer membrane at the interface of the first and last β strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptBFG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptBFG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-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 suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptBFG that is distinct from those of classical ABC transporters that transport substrates across the IM.

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Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty‐acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl‐CoA‐dependent or acyl‐CoA‐independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl‐CoA‐dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane‐bound and soluble forms of DGAT have been identified with very different amino‐acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl‐CoA‐independent pathways via the catalytic action of membrane‐bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty‐acid composition of TAG is also discussed.

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