RESUMEN
Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2-6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.
Asunto(s)
Antibacterianos , Proteínas de la Membrana Bacteriana Externa , Lipopolisacáridos , Proteínas de Transporte de Membrana , Acinetobacter/química , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Viabilidad Microbiana , Conformación Proteica/efectos de los fármacos , Especificidad por SustratoRESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.
Asunto(s)
Antibacterianos , Lipopolisacáridos , Proteínas de Transporte de Membrana , Animales , Humanos , Ratones , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/clasificación , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Desarrollo de MedicamentosRESUMEN
Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria1,2. The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging3. As a result, there are few antibiotic options for P. aeruginosa infections4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely5. Here we assess the whole-cell accumulation of 345 diverse compounds in P. aeruginosa and Escherichia coli. Although certain positively charged compounds permeate both bacterial species, P. aeruginosa is more restrictive compared to E. coli. Computational analysis identified distinct physicochemical properties of small molecules that specifically correlate with P. aeruginosa accumulation, such as formal charge, positive polar surface area and hydrogen bond donor surface area. Mode of uptake studies revealed that most small molecules permeate P. aeruginosa using a porin-independent pathway, thus enabling discovery of general P. aeruginosa accumulation trends with important implications for future antibiotic development. Retrospective antibiotic examples confirmed these trends and these discoveries were then applied to expand the spectrum of activity of a gram-positive-only antibiotic, fusidic acid, into a version that demonstrates a dramatic improvement in antibacterial activity against P. aeruginosa. We anticipate that these discoveries will facilitate the design and development of high-permeating antipseudomonals.
Asunto(s)
Antibacterianos , Diseño de Fármacos , Porinas , Pseudomonas aeruginosa , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Estudios Retrospectivos , Electricidad Estática , Enlace de Hidrógeno , Ácido Fusídico/metabolismo , Diseño de Fármacos/métodosRESUMEN
The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 Å resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8-TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.
Asunto(s)
Modelos Moleculares , Factor de Transcripción TFIID/química , Células Cultivadas , Microscopía por Crioelectrón , Células HeLa , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismoRESUMEN
Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.
Asunto(s)
Baculoviridae/genética , Complejos Multiproteicos/química , Animales , Baculoviridae/metabolismo , Línea Celular , Clonación Molecular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Spodoptera/genética , Spodoptera/metabolismoRESUMEN
The crystal structure of a truncated, soluble quadruple mutant of FtsH from Aquifex aeolicus comprising the AAA and protease domains has been determined at 2.96 Å resolution in space group I222. The protein crystallizes as a hexamer, with the protease domain forming layers in the ab plane. Contacts between these layers are mediated by the AAA domains. These are highly disordered in one crystal form, but are clearly visible in a related form with a shorter c axis. Here, adenosine diphosphate (ADP) is bound to each subunit and the AAA ring exhibits twofold symmetry. The arrangement is different from the ADP-bound state of an analogously truncated, soluble FtsH construct from Thermotoga maritima. The pore is completely closed and the phenylalanine residues in the pore line a contiguous path. The protease hexamer is very similar to those described for other FtsH structures. To resolve certain open issues regarding a conserved glycine in the linker between the AAA and protease domains, as well as the active-site switch ß-strand, mutations have been introduced in the full-length membrane-bound protein. Activity analysis of these point mutants reveals the crucial importance of these residues for proteolytic activity and is in accord with previous interpretation of the active-site switch and the importance of the linker glycine residue.
Asunto(s)
Adenosina Difosfato/química , Bacterias/química , Proteínas Bacterianas/química , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Conformación ProteicaRESUMEN
Polyketide or polyketide-like macrolides (pMLs) continue to serve as a source of inspiration for drug discovery. However, their inherent structural and stereochemical complexity challenges efforts to explore related regions of chemical space more broadly. Here, we report a strategy termed the Targeted Sampling of Natural Product space (TSNaP) that is designed to identify and assess regions of chemical space bounded by this important class of molecules. Using TSNaP, a family of tetrahydrofuran-containing pMLs are computationally assembled from pML inspired building blocks to provide a large collection of natural product-like virtual pMLs. By scoring functional group and volumetric overlap against their natural counterparts, a collection of compounds are prioritized for targeted synthesis. Using a modular and stereoselective synthetic approach, a library of polyketide-like macrolides are prepared to sample these unpopulated regions of pML chemical space. Validation of this TSNaP approach by screening this library against a panel of whole-cell biological assays, reveals hit rates exceeding those typically encountered in small molecule libraries. This study suggests that the TSNaP approach may be more broadly useful for the design of improved chemical libraries for drug discovery.
Asunto(s)
Productos Biológicos , Policétidos , Macrólidos/farmacología , Productos Biológicos/farmacología , Productos Biológicos/química , Descubrimiento de DrogasRESUMEN
The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin had a tenfold higher potency than in synthetic membranes, highlighting the need for native conditions in antibiotics development. The assay is successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of antibiotics targeting BAM.
Asunto(s)
Antibacterianos , Ensayos Analíticos de Alto Rendimiento , Membranas , Antibacterianos/farmacología , Bioensayo , FermentaciónRESUMEN
Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.
RESUMEN
Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.
Asunto(s)
Escherichia coli/fisiología , Familia de Multigenes/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesisRESUMEN
The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA+ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 A resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo- and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.
Asunto(s)
Proteasas ATP-Dependientes/química , Thermotoga maritima/enzimología , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Cartilla de ADN , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Transporte de Proteínas , Especificidad por SustratoRESUMEN
Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.
Asunto(s)
Automatización de Laboratorios/instrumentación , Clonación Molecular/métodos , Complejos Multiproteicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Academias e Institutos , Animales , Baculoviridae , Células Cultivadas , Escherichia coli , Europa (Continente) , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Proteínas Luminiscentes/biosíntesis , Poliproteínas/biosíntesis , Poliproteínas/genética , Ingeniería de Proteínas , SpodopteraRESUMEN
Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.
Asunto(s)
Factor 4A Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/química , ARN Helicasas/química , Proteínas de Saccharomyces cerevisiae/química , Sitios de Unión , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these molecular machines are only present in low amounts in their native hosts, which impede purification from source material. Unraveling their structure and function at high resolution will often depend on heterologous overproduction. Recombinant expression of multiprotein complexes for structural studies can entail considerable, sometimes inhibitory, investment in both labor and materials, in particular if altering and diversifying of the individual subunits are necessary for successful structure determination. Our laboratory has addressed this challenge by developing technologies that streamline the complex production and diversification process. Here, we review several of these developments for recombinant multiprotein complex production using the MultiBac baculovirus/insect cell expression system which we created. We also addressed parallelization and automation of gene assembly for multiprotein complex expression by developing robotic routines for multigene vector generation. In this contribution, we focus on several improvements of baculovirus expression system performance which we introduced: the modifications of the transfer plasmids, the methods for generation of composite multigene baculoviral DNA, and the simplified and standardized expression procedures which we delineated using our MultiBac system.
Asunto(s)
Baculoviridae/genética , Complejos Multiproteicos/genética , Proteínas Recombinantes/genética , Animales , Línea Celular , Clonación Molecular/métodos , Células Eucariotas/metabolismo , Vectores Genéticos/genética , Insectos/citología , Insectos/genética , Complejos Multiproteicos/metabolismo , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Transducción Genética/métodosRESUMEN
All antibiotics have to engage bacterial amphiphilic barriers such as the lipopolysaccharide-rich outer membrane or the phospholipid-based inner membrane in some manner, either by disrupting them outright and/or permeating them and thereby allow the antibiotic to get into bacteria. There is a growing class of cyclic antibiotics, many of which are of bacterial origin, that exhibit activity against Gram-negative bacteria, which constitute an urgent problem in human health. We examine a diverse collection of these cyclic antibiotics, both natural and synthetic, which include bactenecin, polymyxin B, octapeptin, capreomycin, and Kirshenbaum peptoids, in order to identify what they have in common when they interact with bacterial lipid membranes. We find that they virtually all have the ability to induce negative Gaussian curvature (NGC) in bacterial membranes, the type of curvature geometrically required for permeation mechanisms such as pore formation, blebbing, and budding. This is interesting since permeation of membranes is a function usually ascribed to antimicrobial peptides (AMPs) from innate immunity. As prototypical test cases of cyclic antibiotics, we analyzed amino acid sequences of bactenecin, polymyxin B, and capreomycin using our recently developed machine-learning classifier trained on α-helical AMP sequences. Although the original classifier was not trained on cyclic antibiotics, a modified classifier approach correctly predicted that bactenecin and polymyxin B have the ability to induce NGC in membranes, while capreomycin does not. Moreover, the classifier was able to recapitulate empirical structure-activity relationships from alanine scans in polymyxin B surprisingly well. These results suggest that there exists some common ground in the sequence design of hybrid cyclic antibiotics and linear AMPs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/química , Bacterias Gramnegativas/patogenicidad , Humanos , Aprendizaje Automático , Pruebas de Sensibilidad Microbiana , Fosfolípidos/química , Relación Estructura-ActividadRESUMEN
Here, we describe the crystal structures of two distinct isoforms of ligand-free human karyopherin RanBP5 and investigate its global propensity to interact with influenza A virus polymerase. Our results confirm the general architecture and mechanism of the IMB3 karyopherin-ß subfamily whilst also highlighting differences with the yeast orthologue Kap121p. Moreover, our results provide insight into the structural flexibility of ß-importins in the unbound state. Based on docking of a nuclear localisation sequence, point mutations were designed, which suppress influenza PA-PB1 subcomplex binding to RanBP5 in a binary protein complementation assay.
Asunto(s)
Núcleo Celular/metabolismo , Virus de la Influenza A/enzimología , Mutación Puntual , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , beta Carioferinas/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Transporte de Proteínas , beta Carioferinas/genéticaRESUMEN
Many eukaryotic proteins exist in large multisubunit assemblies and often show compromised folding or activity when their interaction partners are not present. Protein complexes in eukaryotes can contain ten or more subunits with individual polypeptides ranging in size up to several hundred kilodalton, severely restricting the application of conventional cloning strategies and imposing constraints on the choice of the expression host. Modern structural molecular biology often depends on introducing diversity into the specimens under investigation, including mutation, truncation and placement of purification aids. Current recombinant expression methods often require a disproportionate labor investment prior to multiprotein expression, and subsequent to expression and analysis do not provide for rapid revision of the experiment. We have developed reagents and protocols for rapid and flexible multiprotein complex expressions suitable for structural biology, focusing on multigene baculoviral vectors and their recombination mediated assembly. A top priority in protein science is automation. Our strategy can be readily adapted in a robotics setup, for baculovirus/insect cell expression of protein complexes, but likewise also for mammalian or prokaryotic hosts.