RESUMO
Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a ß-barrel structure1,2. ß-Barrels are sheets of ß-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 membrane3-5. One subunit of the machines is itself a ß-barrel protein that has a central role in folding other ß-barrels. In Gram-negative bacteria, the ß-barrel assembly machine (BAM) consists of the ß-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)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 ß-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 studies10-12; 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.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/química , Cloroplastos/química , Proteínas de Escherichia coli/química , Bactérias Gram-Negativas/química , Ligação de Hidrogênio , Mitocôndrias/química , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Acinetobacter baumannii/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Lipopolissacarídeos/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/genéticaRESUMO
The ß-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 ß-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.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Cinética , Modelos Moleculares , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de ProteínaRESUMO
The Bam complex assembles ß-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. These proteins comprise cylindrical ß-sheets with long extracellular loops and create pores to allow passage of nutrients and waste products across the membrane. Despite their functional importance, several questions remain about how these proteins are assembled into the OM after their synthesis in the cytoplasm and secretion across the inner membrane. To understand this process better, we studied the assembly of an essential ß-barrel substrate for the Bam complex, BamA. By mutating conserved residues in the ß-barrel domain of this protein, we generated three assembly-defective BamA substrates that stall early in the folding process in the periplasm. Two of the three defective substrates, which harbor mutations within ß-strands, fail to associate productively with the Bam complex. The third substrate, which harbors mutations in a conserved extracellular loop, accumulates on BamD during assembly, but does not integrate efficiently into the membrane. The assembly of all three substrates can be restored by artificially tethering a region of the substrate, which ultimately becomes an extracellular loop, to the lumen of the forming ß-barrel. These results imply that a critical step in the folding process involves the interaction of residues on the interior of the nascent ß-barrel wall with residues in one of the extracellular loops. We conclude that a prerequisite for membrane integration of ß-barrel proteins is burial of the extracellular loops within the forming ß-barrel.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Periplasma/química , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Membranas/química , Membranas/metabolismo , Complexos Multiproteicos/química , Periplasma/metabolismo , Conformação Proteica em Folha beta , Especificidade por SubstratoRESUMO
The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts contain ß-barrel integral membrane proteins. In bacteria, the five-protein ß-barrel assembly machine (Bam) accelerates the folding and membrane integration of these proteins. The central component of the machine, BamA, contains a ß-barrel domain that can adopt a lateral-open state with its N-terminal and C-terminal ß-strands unpaired. Recently, strategies have been developed to capture ß-barrel folding intermediates on the Bam complex. Biochemical and structural studies provide support for a model in which substrates assemble at the lateral opening of BamA. In this model, the N-terminal ß-strand of BamA captures the C-terminal ß-strand of substrates by hydrogen bonding to allow their directional folding and subsequent release into the membrane.
Assuntos
Proteínas de Escherichia coli , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bactérias Gram-Negativas , Dobramento de ProteínaRESUMO
The ß-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane ß-barrel proteins. BamA, an essential component of the complex, is itself a ß-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 ß-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 ß-barrel. Importantly, we identify contacts between the assembling ß-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 ß-barrel assembly.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Motivos de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Membrana Celular , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0 Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.
Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glicosiltransferases/metabolismo , Synechocystis/enzimologia , Animais , Animais Geneticamente Modificados , Glicosiltransferases/genética , Humanos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Modelos Moleculares , Conformação Proteica , Peixe-ZebraRESUMO
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.
Assuntos
Arabinose/análogos & derivados , Proteínas de Bactérias/química , Cupriavidus/enzimologia , Lipídeo A/química , Pentosiltransferases/química , Amino Açúcares/química , Arabinose/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glicosilação , Mutagênese , Mutação , Pentosiltransferases/genética , Pentosiltransferases/ultraestrutura , Fosfatos de Poli-Isoprenil/química , Polimixinas/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas de Bactérias/química , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/química , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/metabolismo , Micrococcaceae/enzimologia , Fosfatos de Fosfatidilinositol/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/genética , Cristalografia por Raios X , Cinética , Micrococcaceae/química , Micrococcaceae/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologiaRESUMO
The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyses the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalysed solely by CDP-APs. Here we report the 2.0 Å resolution crystal structure of a representative CDP-AP from Archaeoglobus fulgidus. The enzyme (AF2299) is a homodimer, with each protomer consisting of six transmembrane helices and an N-terminal cytosolic domain. A polar cavity within the membrane accommodates the active site, lined with the residues from an absolutely conserved CDP-AP signature motif (D(1)xxD(2)G(1)xxAR...G(2)xxxD(3)xxxD(4)). Structures in the apo, CMP-bound, CDP-bound and CDP-glycerol-bound states define functional roles for each of these eight conserved residues and allow us to propose a sequential, base-catalysed mechanism universal for CDP-APs, in which the fourth aspartate (D4) acts as the catalytic base.