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Author(s): David Tomasek [sup.1] [sup.2] , Shaun Rawson [sup.3] , James Lee [sup.1] [sup.2] [sup.5] , Joseph S. Wzorek [sup.2] [sup.6] , Stephen C. Harrison [sup.3] [sup.4] , Zongli Li [...], Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a [beta]-barrel structure.sup.1,2. [beta]-Barrels are sheets of [beta]-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane.sup.3-5. One subunit of the machines is itself a [beta]-barrel protein that has a central role in folding other [beta]-barrels. In Gram-negative bacteria, the [beta]-barrel assembly machine (BAM) consists of the [beta]-barrel protein BamA, and four lipoproteins.sup.5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP).sup.9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid [beta]-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies.sup.10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete. Cryo-electron microscopy structures of a folding intermediate on the BAM complex of Escherichia coli reveal how interactions between the BamA catalyst and substrate permit stable association during folding, followed by rapid turnover.

Author(s): Tristan W. Owens [sup.1] , Rebecca J. Taylor [sup.1] , Karanbir S. Pahil [sup.1] , Blake R. Bertani [sup.2] , Natividad Ruiz [sup.2] , Andrew C. Kruse [sup.3] , [...], Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide.sup.1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface.sup.1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown.sup.2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient. Crystal structures of a five-protein complex comprising the inner-membrane components of the bacterial lipopolysaccharide transporter provide insight into the mechanism of extraction of lipopolysaccharide from the inner membrane and its transport to the outer membrane.

Author(s): Megan Sjodt [1]; Kelly Brock [2]; Genevieve Dobihal [3]; Patricia D. A. Rohs [3]; Anna G. Green [2]; Thomas A. Hopf [2]; Alexander J. Meeske [3]; Veerasak Srisuknimit [4]; [...]

Author(s): Alexander J. Meeske [1]; Eammon P. Riley [1]; William P. Robins [1]; Tsuyoshi Uehara [1]; John J. Mekalanos [1]; Daniel Kahne [2, 3]; Suzanne Walker [1, 2, 3]; Andrew [...], Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.