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.

Deposits of amyloid fibrils of α-synuclein are the histological hallmarks of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, with hereditary mutations in αsynuclein linked to the first two of these conditions. Seeing the changes to the structures of amyloid fibrils bearing these mutations may help to understand these diseases. To this end, we determined the cryo-EM structures of α-synuclein fibrils containing the H50Q hereditary mutation. We find that the H50Q mutation results in two previously unobserved polymorphs of αsynuclein: Narrow and Wide Fibrils, formed from either one or two protofilaments, respectively. These structures recapitulate conserved features of the wild-type fold but reveal new structural elements including a previously unobserved hydrogen bond network and surprising new protofilament arrangements. The structures of the H50Q polymorphs help to rationalize the faster aggregation kinetics, higher seeding capacity in biosensor cells, and greater cytotoxicity we observe for H50Q compared to wild-type α-synuclein.

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.

Aggregation of α-synuclein is a defining molecular feature of Parkinson’s disease, Lewy body dementia, and multiple systems atrophy. Hereditary mutations in α-synuclein are linked to both Parkinson’s disease and Lewy body dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy body dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on α-synuclein fibril structure is fundamental to α-synuclein biology. We therefore determined the cryo-electron microscopy (cryo-EM) structure of α-synuclein fibrils containing the hereditary E46K mutation. The 2.5-Å structure reveals a symmetric double protofilament in which the molecules adopt a vastly rearranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which form the E46-K80 salt bridge in the wild-type fibril structure. We hypothesize that, under our conditions, the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts α-synuclein into a kinetic trap—a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure.

Significance Statement:Parkinson's is the second most prevalent neurodegenerative condition, leading to movement disorders, and dementia in some cases. Because of the strong association of this condition with amyloid aggregates of the protein α-synuclein, structural understanding of these amyloid aggregates may be the path to eventual therapies. Our study of the structure of a variant αsynuclein inherited in families afflicted with a clinical picture of parkinsonism and Lewy Body Dementia supplements recent structures of the wild type structure, and shows how a single residue change can result in a greatly changed structure that may underlie the inherited form of the disease. Abstract:Aggregation of α-synuclein is a defining molecular feature of Parkinson's disease, Lewy Body Dementia, and Multiple Systems Atrophy. Hereditary mutations in α-synuclein are linked to both Parkinson's disease and Lewy Body Dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy Body Dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on α-synuclein fibril structure is fundamental to α-synuclein biology. We therefore determined the cryoEM structure of α-synuclein fibrils containing the hereditary E46K mutation. The 2.5 Å structure reveals a symmetric double protofilament in which the molecules adopt a vastly rearranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which forms the E46-K80 salt-bridge in the wild-type fibril structure. We hypothesize that under our conditions the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts α-synuclein into a kinetic trap -a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure. Introduction:The group of diseases termed the synucleinopathies -Parkinson's Disease (PD), Lewy Body Dementia (LBD), and Multiple Systems Atrophy (MSA) -is thought to be caused by the aggregation of α-synuclein (α-syn) into amyloid fibrils. The causal relationship between the formation of amyloid fibrils of α-syn and the synucleinopathies is supported by several observations. Aggregated α-syn is a major component of Lewy Bodies, the hallmark lesion in PD and LBD, and the hallmark lesions of MSA 1,2 . Hereditary mutations in α-syn are linked to familial forms of PD and LBD 3 . Overexpression of wild-type α-syn via dominantly inherited duplications and triplications of the gene that encodes α-syn, SNCA, are sufficient to cause PD 4-6 . Further, the injection of fibrils of α-syn into the brains of mice induced PD-like pathology including Lewy body and Lewy neurite formation, cell-to-cell spreading of Lewy body pathology, and motor deficits similar to PD 8 . Although it is never fully possible to establish causation, these combined observations suggest the c...