RESUMO
The V27A mutation confers adamantane resistance on the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to determine structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to that of the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics simulation of the M2(22-46) V27A-spiro-adamantyl amine complex predicts with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2(22-46) V27A complex.
Assuntos
Adamantano/química , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/ultraestrutura , Adamantano/análogos & derivados , Adamantano/farmacologia , Aminas/metabolismo , Antivirais/farmacologia , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Farmacorresistência Bacteriana/genética , Farmacorresistência Viral/efeitos dos fármacos , Humanos , Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Mutação/genética , Radiografia/métodos , Proteínas da Matriz Viral/genéticaRESUMO
POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Peptídeos/metabolismo , Prótons , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Xanthomonas/química , Xanthomonas/metabolismoRESUMO
Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.
Assuntos
Proteínas de Bactérias/química , Membrana Celular/metabolismo , Diacilglicerol Quinase/química , Diacilglicerol Quinase/metabolismo , Proteínas de Membrana/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Diacilglicerol Quinase/genética , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Lipídeos , Magnésio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Zinco/farmacologiaRESUMO
Label-free differential scanning fluorimetry (DSF) is a relatively new method for evaluating the stability of proteins. It can be used as a screening tool for downstream applications such as crystallization. The method is attractive in that it requires miniscule quantities of proteins, it can be performed using intrinsic tryptophan and tyrosine fluorescence, and, with the right equipment, it is easy to perform. To date, the method has been used with proteins in liquid solutions and dispersions. It was of interest to determine if DSF could be used with membrane proteins in the lipid cubic phase (LCP), which increasingly is being used for crystallization in support of structure-function studies. The cubic phase is viscous. Furthermore, in coexistence with excess aqueous solution, as happens during crystallization trials, it can become turbid and scatter light. The concern was that these features may render the mesophase unsuitable for DSF analysis. However, using lysozyme and four integral membrane proteins we demonstrate that the method works with all tested proteins in solution and in the LCP. Of note is the observation that some of the test membrane proteins are more stable while others are less so in the mesophase. The method also works in ligand binding measurements. Thus, DSF should prove useful as an analytical tool for identifying host and additive lipids, detergents, precipitants and chemical probes that support the generation of quality crystals by the cubic phase method. Microscale thermophoresis was used to supplement the DSF study and was also shown to work with proteins in the mesophase. Measurements with lysozyme highlight the utility of the cubic mesophase as a model system in which to perform confinement studies.
Assuntos
Fluorometria , Lipídeos/química , Proteínas de Membrana/química , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Galinhas , Escherichia coli/química , Muramidase/química , Estabilidade Proteica , Pseudomonas aeruginosa/química , Solubilidade , TemperaturaRESUMO
We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Membrana/química , Complexos Multiproteicos/química , Difração de Raios X/métodos , Animais , Humanos , Modelos Moleculares , Conformação Proteica , Software , SíncrotronsRESUMO
Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.
Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos a3/metabolismo , Citocromos a/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Thermus thermophilus/enzimologia , Azurina/metabolismo , Domínio Catalítico , Membrana Celular/metabolismo , Cristalização , Cristalografia por Raios X , Transporte de Elétrons , Elétrons , Glicerofosfolipídeos/química , Glicerofosfolipídeos/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Prótons , Água/química , Água/metabolismoRESUMO
Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.
Assuntos
Bacillus subtilis/enzimologia , Membrana Celular/enzimologia , Fosfatidato Fosfatase/química , Fosfatidato Fosfatase/metabolismo , Bacillus subtilis/genética , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/metabolismo , Genes Bacterianos , Teste de Complementação Genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfatidato Fosfatase/genética , Fosfatidilgliceróis/metabolismo , Solubilidade , Especificidade por SubstratoRESUMO
G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human ß(2) adrenergic receptor (ß(2)AR) as a guide, we designed a ß(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent ß(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound ß(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Cristalização , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Agonismo Inverso de Drogas , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Procaterol/química , Procaterol/metabolismo , Propanolaminas/química , Propanolaminas/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ß(2) adrenergic receptor (ß(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ß(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ß(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ß(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animais , Domínio Catalítico , Bovinos , Cristalização , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , RatosRESUMO
We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.
Assuntos
Detergentes/química , Lipídeos/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Micelas , Diacilglicerol Quinase/química , Difusão , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Fluorescência Verde/química , Halobacteriaceae/química , Espectroscopia de Ressonância Magnética/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas de Transporte de Monossacarídeos/química , Nanopartículas/química , Plasmídeos/metabolismo , Rodopsinas Sensoriais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Simportadores/químicaRESUMO
An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.
Assuntos
Proteínas de Bactérias/química , Streptococcus thermophilus , Simportadores/química , Sítios de Ligação , Cristalografia por Raios X , Dipeptídeos/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Oligopeptídeos/química , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.
Assuntos
Proteínas de Membrana/química , Cristalização , Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Escherichia coli/enzimologia , Conformação ProteicaRESUMO
The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the ß2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.
Assuntos
Cristalografia por Raios X/métodos , Proteínas de Membrana/química , Conformação ProteicaRESUMO
Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.
Assuntos
Membrana Celular/enzimologia , Diacilglicerol Quinase/química , Proteínas de Membrana/química , Conformação Proteica , Sistema Livre de Células , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaios Enzimáticos , Regulação Enzimológica da Expressão Gênica , Lipídeos/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Multimerização ProteicaRESUMO
The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common ß-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.
Assuntos
Pseudomonas aeruginosa/metabolismo , Alginatos/química , Sequência de Bases , Membrana Celular/metabolismo , Cristalografia por Raios X , Primers do DNA , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein-protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.
Assuntos
Cristalização/métodos , Lipídeos/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Difração de Raios XRESUMO
The lipidic cubic mesophase has been used to crystallize important membrane proteins for high-resolution structure determination. To date, however, no integral membrane enzymes have yielded to this method, the in meso. For a crystal structure to be meaningful the target protein must be functional. Using the in meso method with a membrane enzyme requires that the protein is active in the mesophase that grows crystals. Because the cubic phase is sticky and viscous and is bicontinuous topologically, quantitatively assessing enzyme activity in meso is a challenge. Here, we describe a procedure for characterizing the catalytic properties of the integral membrane enzyme, diacylglycerol kinase, reconstituted into the bilayer of the lipidic cubic phase. The kinase activity of this elusive crystallographic target was monitored spectrophotometrically using a coupled assay in a high-throughput, 96-well plate format. In meso, the enzyme exhibits classic Michaelis-Menten kinetics and works with a range of lipid substrates. The fact that the enzyme and its lipid substrate and product remain confined to the porous mesophase while its water-soluble substrate and product are free to partition into the aqueous bathing solution suggests a general and convenient approach for characterizing membrane enzymes that function with lipids in a membrane-like environment. The distinctive rheology of the cubic phase means that a procedural step to physically separate substrate from product is not needed. Because of its open, bicontinuous nature, the cubic phase offers the added benefit that the protein is accessible for assay from both sides of the membrane.
Assuntos
Enzimas/química , Lipídeos/química , Proteínas de Membrana/química , Modelos Biológicos , Catálise , Cristalização , Cristalografia por Raios X , Diacilglicerol Quinase/química , Bicamadas Lipídicas , Membranas ArtificiaisRESUMO
A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature-composition phase diagrams for 7.10 MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG's chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane ß-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid.
RESUMO
There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.
Assuntos
Antibacterianos , Desenho de Fármacos , Peptídeos Cíclicos , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Testes de Sensibilidade Microbiana , Depsipeptídeos/farmacologia , Depsipeptídeos/química , Lipoproteínas/química , Lipoproteínas/metabolismo , Lipoproteínas/farmacologia , Lipoproteínas/antagonistas & inibidores , Proteínas de Bactérias , Peptídeos , Ácido Aspártico EndopeptidasesRESUMO
Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.