Mitochondria, chloroplasts, and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure 1 , 2 . β-barrels are sheets of β-strands wrapped into a cylinder with the first strand hydrogen-bonded to the last strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane 3 – 5 . One subunit of the machines is itself a β-barrel protein that plays a central role in folding other β-barrels. In Gram-negative bacteria, the β- b arrel a ssembly m achine (Bam) consists of the β-barrel protein BamA and four lipoproteins 5 – 8 . To understand how the Bam complex accelerates folding without using exogenous energy (e.g., ATP) 9 , we trapped folding intermediates on the machine. We report here the structure of the Bam complex folding BamA itself. The BamA catalyst (BamA M , for BamA machine ) forms an asymmetric hybrid β-barrel with the BamA substrate (BamA S ). The N-terminal edge of BamA M has an antiparallel hydrogen-bonded interface with the C-terminal edge of BamA S , consistent with previous crosslinking studies 10 – 12 ; the other edges of BamA M and BamA S 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 once folding has finished. Features at each end of the substrate overcome the 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.

Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-L-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

The β-barrel assembly machine (Bam) complex folds and inserts integral membrane proteins into the outer membrane of Gram-negative bacteria. The two essential components of the complex, BamA and BamD, both interact with substrates, but how the two coordinate with each other during assembly is not clear. To elucidate aspects of this process we slowed the assembly of an essential β-barrel substrate of the Bam complex, LptD, by changing a conserved residue near the C terminus. This defective substrate is recruited to the Bam complex via BamD but is unable to integrate into the membrane efficiently. Changes in the extracellular loops of BamA partially restore assembly kinetics, implying that BamA fails to engage this defective substrate. We conclude that substrate binding to BamD activates BamA by regulating extracellular loop interactions for folding and membrane integration.

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.