RESUMO
Cholesterol-dependent cytolysins (CDCs) comprise a large family of pore-forming toxins produced by Gram-positive bacteria, which are used to attack eukaryotic cells. Here, we functionally characterize a family of 2-component CDC-like (CDCL) toxins produced by the Gram-negative Bacteroidota that form pores by a mechanism only described for the mammalian complement membrane attack complex (MAC). We further show that the Bacteroides CDCLs are not eukaryotic cell toxins like the CDCs, but instead bind to and are proteolytically activated on the surface of closely related species, resulting in pore formation and cell death. The CDCL-producing Bacteroides is protected from the effects of its own CDCL by the presence of a surface lipoprotein that blocks CDCL pore formation. These studies suggest a prevalent mode of bacterial antagonism by a family of two-component CDCLs that function like mammalian MAC and that are wide-spread in the gut microbiota of diverse human populations.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento , Humanos , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Citotoxinas/metabolismo , Microbioma Gastrointestinal , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/imunologia , Animais , Células Eucarióticas/metabolismoRESUMO
Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.
Assuntos
Antimaláricos , Aspartato-tRNA Ligase , Animais , Humanos , Plasmodium falciparum/genética , Asparagina/metabolismo , Aspartato-tRNA Ligase/genética , Aminoacil-RNA de Transferência/metabolismo , Antimaláricos/farmacologia , Mamíferos/genéticaRESUMO
Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family ß-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.
Assuntos
Citocinas , Interleucina-11 , Humanos , Interleucina-11/genética , Receptor gp130 de Citocina/genética , Interleucina-6/metabolismo , Antígenos CD/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina-6/metabolismoRESUMO
Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.
RESUMO
C-reactive protein (CRP) is an early-stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro-inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X-ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide in vitro and in vivo proof-of-concept data demonstrating that the low molecular weight tool compound inhibits CRP-driven exacerbation of local inflammatory responses, while potentially preserving pathogen-defense functions of CRP. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy.
Assuntos
Proteína C-Reativa , Fosforilcolina , Humanos , Fosforilcolina/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Membrana Celular/metabolismo , Anti-InflamatóriosRESUMO
SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated samples that can be adapted for the detection of viruses in clinical studies.
Assuntos
Células Epiteliais , Heparina , SARS-CoV-2 , Replicação Viral , Humanos , COVID-19 , Células Epiteliais/virologia , Heparina/farmacologia , Pandemias , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.
Assuntos
COVID-19 , Trombose , Tromboembolia Venosa , Biomarcadores , Proteína C-Reativa/metabolismo , Humanos , Inflamação/metabolismo , Fosforilcolina , Isoformas de Proteínas/metabolismo , TromboinflamaçãoRESUMO
The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
Assuntos
Toxinas Bacterianas , Proteínas Hemolisinas , Animais , Toxinas Bacterianas/metabolismo , Clostridium perfringens/metabolismo , Proteínas Hemolisinas/metabolismo , Lipossomos/metabolismo , Mamíferos/metabolismoRESUMO
The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.
Assuntos
Toxinas Bacterianas , Citotoxinas , Citotoxinas/metabolismo , Toxinas Bacterianas/genética , Colesterol/metabolismo , Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Assuntos
Antimaláricos , Malária Falciparum , Terapia de Alvo Molecular , Plasmodium falciparum , Biossíntese de Proteínas , Proteínas de Protozoários , Tirosina-tRNA Ligase , Adenosina/análogos & derivados , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cristalografia por Raios X , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Ácidos Sulfônicos/química , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismoRESUMO
Alzheimer's disease is a common and devastating age-related disease with no effective disease-modifying treatments. Human genetics has implicated a wide range of cell surface receptors as playing a role in the disease, many of which are involved in the production or clearance of neurotoxins in the brain. Amyloid precursor protein, a membrane-bound signaling molecule, is at the very heart of the disease: hereditary mutations in its gene are associated with a greatly increased risk of getting the disease. A proteolytic breakdown product of amyloid precursor protein, the neurotoxic Aß peptide, has been the target for many drug discovery efforts. Antibodies have been designed to target Aß production with some success, although they have not proved efficacious in clinical trials with regards to cognitive benefits to date. Many of the recently identified genes associated with late-onset Alzheimer's disease risk are integral to the innate immune system. Some of these genes code for microglial proteins, such as the strongest genetic risk factor for the disease, namely APOE, and the cell surface receptors CD33 and TREM2 which are involved in clearance of the Aß peptide from the brain. In this review, we show how structural biology has provided key insights into the normal functioning of these cell surface receptors and provided a framework for developing novel treatments to combat Alzheimer's disease.
RESUMO
The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
Assuntos
Compostos de Boro/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Administração Oral , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Domínio Catalítico , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Plasmodium falciparum/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/químicaRESUMO
A common form of cellular attack by pathogenic bacteria is to secrete pore-forming toxins (PFTs). Capable of forming transmembrane pores in various biological membranes, PFTs have also been identified in a diverse range of other organisms such as sea anemones, earthworms and even mushrooms and trees. The mechanism of pore formation by PFTs is associated with substantial conformational changes in going from the water-soluble to transmembrane states of the protein. The determination of the crystal structures for numerous PFTs has shed much light on our understanding of these proteins. Other than elucidating the atomic structural details of PFTs and the conformational changes that must occur for pore formation, crystal structures have revealed structural homology that has led to the discovery of new PFTs and new PFT families. Here we review some key crystallographic results together with complimentary approaches for studying PFTs. We discuss how these studies have impacted our understanding of PFT function and guided research into biotechnical applications.
Assuntos
Toxinas Bacterianas , Membrana Celular , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas Citotóxicas Formadoras de PorosRESUMO
BACKGROUND: Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6 and IL-11 and variable oncostatin M (OSM) and IL-27 levels but sparing leukemia inhibitory factor (LIF) signaling. OBJECTIVE: Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants. METHODS: We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed. RESULTS: We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6- and IL-11-dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane-proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome. CONCLUSION: Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants.
Assuntos
Receptor gp130 de Citocina , Síndrome de Job , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Criança , Receptor gp130 de Citocina/química , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/imunologia , Citocinas/genética , Citocinas/imunologia , Genes Recessivos , Humanos , Síndrome de Job/genética , Síndrome de Job/imunologia , Masculino , RNA-Seq , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Sequenciamento do ExomaRESUMO
The cholesterol-dependent cytolysins (CDCs) are bacterial, ß-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.IMPORTANCE The cholesterol-dependent cytolysins' pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the ß-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.
Assuntos
Motivos de Aminoácidos , Colesterol/metabolismo , Citotoxinas/química , Flavobacteriaceae/química , Animais , Membrana Celular/metabolismo , Cristalografia por Raios X , Culicidae/microbiologia , Citotoxinas/genética , Flavobacteriaceae/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMO
Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the ß-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11-IL-11Rα complex.
Assuntos
Subunidade alfa de Receptor de Interleucina-11/química , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Interleucina-11/metabolismo , Área Sob a Curva , Linhagem Celular Tumoral , Entropia , Humanos , Subunidade alfa de Receptor de Interleucina-11/genética , Modelos Moleculares , Mutação/genética , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Assuntos
Plasmodium falciparum/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Artemisininas/farmacologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Multimerização Proteica , Proteostase , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Most structure-based drug discovery methods utilize crystal structures of receptor proteins. Crystal engineering, on the other hand, utilizes the wealth of chemical information inherent in small-molecule crystal structures in the Cambridge Structural Database (CSD). We show that the interaction surfaces and shapes of molecules in experimentally determined small-molecule crystal structures can serve as effective tools in drug discovery. Our description of the shape and interaction propensities of molecules in their crystal structures can be used to screen them for specific binding compatibility with protein targets, as demonstrated through the high-throughput profiling of around 138 000 small-molecule structures in the CSD and a series of drug-protein crystal structures. Electron-density-based intermolecular boundary surfaces in small-molecule crystal structures and in target-protein pockets are utilized to identify potential ligand molecules from the CSD based on 3D shape and intermolecular interaction matching.
Assuntos
Bases de Dados Factuais , Descoberta de Drogas , Conformação Proteica , Proteínas/química , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Relação Quantitativa Estrutura-AtividadeRESUMO
The crystal structures of the soluble monomers of the pore-forming cholesterol-dependent cytolysins (CDCs) contain two α-helical bundles that flank a twisted core ß-sheet. This protein fold is the hallmark of the CDCs, as well as of the membrane attack complex/perforin immune defense proteins and the stonefish toxins. To form the ß-barrel pore, a core ß-sheet is flattened to align the membrane-spanning ß-hairpins. Concomitantly with this conformational change, the two α-helical bundles that flank the core ß-sheet break their restraining contacts and refold into two membrane-spanning ß-hairpins of the ß-barrel pore. The studies herein show that in the monomer structure of the archetype CDC perfringolysin O (PFO), a conserved Met-Met-Phe triad simultaneously contributes to maintaining the twist in this core ß-sheet, as well as restricting the α-helical-to-ß-strand transition necessary to form one of two membrane-spanning ß-hairpins. A previously identified intermolecular π-stacking interaction is now shown to disrupt the interactions mediated by this conserved triad. This is required to establish the subsequent intermolecular electrostatic interaction, which has previously been shown to drive the final conformational changes necessary to form the ß-barrel pore. Hence, these studies show that the intermolecular π-stacking and electrostatic interactions work in tandem to flatten the core ß-sheet and initiate the α-helical-to-ß-strand transitions to form the ß-barrel pore.IMPORTANCE A unique feature of the CDC/MACPF/SNTX (cholesterol-dependent cytolysin/membrane attack complex perforin/stonefish toxin) superfamily of pore-forming toxins is that the ß-strands that comprise the ß-barrel pore are derived from a pair of α-helical bundles. These studies reveal the molecular basis by which the formation of intermolecular interactions within the prepore complex drive the disruption of intramolecular interactions within each monomer of the prepore to trigger the α-helical-to-ß-strand transition and formation of the ß-barrel pore.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Cristalografia por Raios X , Ligação Proteica , Conformação ProteicaRESUMO
The cholesterol-dependent cytolysins (CDCs) are a family of bacterial protein toxins specifically targeting eukaryotic cells through the absolute requirement for high concentrations of cholesterol in the target cells' lipid membrane. The soluble monomeric protein secreted by the bacteria oligomerizes on the surface of the target cell, and the complex formed then undergoes a concerted structural transition that results in the creation of a multimeric protein pore. Recognition of the cholesterol-rich membrane by CDCs is a surprisingly subtle process that takes place at the interface between the membrane and surrounding aqueous environment. The structure and composition of the lipid membrane modulates the efficiency with which the protein can identify cholesterol and alters the concentration of sterol required for membrane binding. Some of the details of the interplay between protein and membrane remain to be resolved, and in this review we present a current perspective on CDC pore formation, with particular focus on the role of the lipid bilayer and cholesterol accessibility.