RÉSUMÉ
BACKGROUND: Understanding the epidemiology of carbapenem-resistant A. baumannii complex (CRAB) and the patients impacted is an important step toward informing better infection prevention and control practices and improving public health response. METHODS: Active, population-based surveillance was conducted for CRAB in 9 U.S. sites from January 1 to December 31, 2019. Medical records were reviewed, isolates were collected and characterized including antimicrobial susceptibility testing and whole genome sequencing. RESULTS: Among 136 incident cases in 2019, 66 isolates were collected and characterized; 56.5% were from cases who were male, 54.5% were from persons of Black or African American race with non-Hispanic ethnicity, and the median age was 63.5 years. Most isolates, 77.2%, were isolated from urine, and 50.0% were collected in the outpatient setting; 72.7% of isolates harbored an acquired carbapenemase gene (aCP), predominantly blaOXA-23 or blaOXA-24/40; however, an isolate with blaNDM was identified. The antimicrobial agent with the most in vitro activity was cefiderocol (96.9% of isolates were susceptible). CONCLUSIONS: Our surveillance found that CRAB isolates in the U.S. commonly harbor an aCP, have an antimicrobial susceptibility profile that is defined as difficult-to-treat resistance, and epidemiologically are similar regardless of the presence of an aCP.
Sujet(s)
Infections à Acinetobacter , Acinetobacter baumannii , Antibactériens , Carbapénèmes , Tests de sensibilité microbienne , Humains , Acinetobacter baumannii/effets des médicaments et des substances chimiques , Acinetobacter baumannii/génétique , Acinetobacter baumannii/isolement et purification , États-Unis/épidémiologie , Infections à Acinetobacter/épidémiologie , Infections à Acinetobacter/microbiologie , Mâle , Carbapénèmes/pharmacologie , Adulte d'âge moyen , Sujet âgé , Femelle , Antibactériens/pharmacologie , Adulte , Sujet âgé de 80 ans ou plus , Séquençage du génome entier , bêta-Lactamases/génétique , Maladies transmissibles émergentes/microbiologie , Maladies transmissibles émergentes/épidémiologie , Jeune adulte , Protéines bactériennes/génétiqueRÉSUMÉ
Recent advances in AI-based methods have revolutionized the field of structural biology. Concomitantly, high-throughput sequencing and functional genomics technologies have enabled the detection and generation of variants at an unprecedented scale. However, efficient tools and resources are needed to link these two disparate data types - to "map" variants onto protein structures, to better understand how the variation causes disease and thereby design therapeutics. Here we present the Genomics 2 Proteins Portal (G2P; g2p.broadinstitute.org/): a human proteome-wide resource that maps 19,996,443 genetic variants onto 42,413 protein sequences and 77,923 structures, with a comprehensive set of structural and functional features. Additionally, the G2P portal generalizes the capability of linking genomics to proteins beyond databases by allowing users to interactively upload protein residue-wise annotations (variants, scores, etc.) as well as the protein structure to establish the connection. The portal serves as an easy-to-use discovery tool for researchers and scientists to hypothesize the structure-function relationship between natural or synthetic variations and their molecular phenotype.
RÉSUMÉ
The preclinical time course of SARS-CoV-2 shedding is not well-described. Understanding this time course will help to inform risk of SARS-CoV-2 transmission. During an outbreak in a congregate setting, we collected paired mid-turbinate nasal swabs for antigen testing and reverse-transcription polymerase chain reaction (RT-PCR) every other day from all consenting infected and exposed persons. Among 12 persons tested prospectively before and during SARS-CoV-2 infection, ten of 12 participants (83%) had completed a primary COVID-19 vaccination series prior to the outbreak. We recovered SARS-CoV-2 in viral culture from 9/12 (75%) of participants. All three persons from whom we did not recover SARS-CoV-2 in viral culture had completed their primary vaccination series. We recovered SARS-CoV-2 from viral culture in 6/9 vaccinated persons and before symptom onset in 3/6 symptomatic persons. These findings underscore the need for both non-pharmaceutical interventions and vaccination to mitigate transmission.
Sujet(s)
COVID-19 , SARS-CoV-2 , Humains , COVID-19/prévention et contrôle , Excrétion virale , Vaccins contre la COVID-19 , Dépistage de la COVID-19RÉSUMÉ
Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
Sujet(s)
Cryomicroscopie électronique , Protéines et peptides de signalisation intracellulaire , Complexes multiprotéiques , Protein Phosphatase 1 , Protéines G ras , Motifs d'acides aminés , Sites de fixation , Guanosine triphosphate/métabolisme , Humains , Protéines et peptides de signalisation intracellulaire/composition chimique , Protéines et peptides de signalisation intracellulaire/génétique , Protéines et peptides de signalisation intracellulaire/métabolisme , Système de signalisation des MAP kinases , Complexes multiprotéiques/composition chimique , Complexes multiprotéiques/métabolisme , Complexes multiprotéiques/ultrastructure , Mutation faux-sens , Phosphorylation , Liaison aux protéines , Protein Phosphatase 1/composition chimique , Protein Phosphatase 1/métabolisme , Protein Phosphatase 1/ultrastructure , Stabilité protéique , Kinases raf , Protéines G ras/composition chimique , Protéines G ras/métabolisme , Protéines G ras/ultrastructureRÉSUMÉ
Background: We estimated SARS-CoV-2 Delta- and Omicron-specific effectiveness of two and three mRNA COVID-19 vaccine doses in adults against symptomatic illness in US outpatient settings. Methods: Between October 1, 2021, and February 12, 2022, research staff consented and enrolled eligible participants who had fever, cough, or loss of taste or smell and sought outpatient medical care or clinical SARS-CoV-2 testing within 10 days of illness onset. Using the test-negative design, we compared the odds of receiving two or three mRNA COVID-19 vaccine doses among SARS-CoV-2 cases versus controls using logistic regression. Regression models were adjusted for study site, age, onset week, and prior SARS-CoV-2 infection. Vaccine effectiveness (VE) was calculated as (1 - adjusted odds ratio) × 100%. Results: Among 3847 participants included for analysis, 574 (32%) of 1775 tested positive for SARS-CoV-2 during the Delta predominant period and 1006 (56%) of 1794 participants tested positive during the Omicron predominant period. When Delta predominated, VE against symptomatic illness in outpatient settings was 63% (95% CI: 51% to 72%) among mRNA two-dose recipients and 96% (95% CI: 93% to 98%) for three-dose recipients. When Omicron predominated, VE was 21% (95% CI: -6% to 41%) among two-dose recipients and 62% (95% CI: 48% to 72%) among three-dose recipients. Conclusions: In this adult population, three mRNA COVID-19 vaccine doses provided substantial protection against symptomatic illness in outpatient settings when the Omicron variant became the predominant cause of COVID-19 in the United States. These findings support the recommendation for a third mRNA COVID-19 vaccine dose.
Sujet(s)
COVID-19 , Patients en consultation externe , Adulte , Humains , Dépistage de la COVID-19 , Vaccins contre la COVID-19 , COVID-19/prévention et contrôle , SARS-CoV-2/génétique , ARN messager/génétiqueRÉSUMÉ
Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.
Sujet(s)
Anti-inflammatoires/pharmacologie , Arthrite expérimentale/traitement médicamenteux , Multimérisation de protéines/effets des médicaments et des substances chimiques , Transduction du signal/effets des médicaments et des substances chimiques , Facteur de nécrose tumorale alpha/antagonistes et inhibiteurs , Animaux , Anti-inflammatoires/usage thérapeutique , Arthrite expérimentale/immunologie , Lignée cellulaire , Cristallographie aux rayons X , Découverte de médicament , Mâle , Souris , Simulation de dynamique moléculaire , Infiltration par les neutrophiles/effets des médicaments et des substances chimiques , Granulocytes neutrophiles/effets des médicaments et des substances chimiques , Granulocytes neutrophiles/immunologie , Stabilité protéique/effets des médicaments et des substances chimiques , Structure quaternaire des protéines/effets des médicaments et des substances chimiques , Récepteur au facteur de nécrose tumorale de type I/immunologie , Récepteur au facteur de nécrose tumorale de type I/métabolisme , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Protéines recombinantes/ultrastructure , Transduction du signal/immunologie , Relation structure-activité , Résultat thérapeutique , Facteur de nécrose tumorale alpha/immunologie , Facteur de nécrose tumorale alpha/isolement et purification , Facteur de nécrose tumorale alpha/métabolisme , Facteur de nécrose tumorale alpha/ultrastructureRÉSUMÉ
Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84%) missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology.
Sujet(s)
Mitogen-Activated Protein Kinase 1/génétique , Mutation faux-sens/génétique , Lignée cellulaire tumorale , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Dual Specificity Phosphatase 6/métabolisme , Humains , Modèles moléculaires , Phénotype , Phosphorylation/effets des médicaments et des substances chimiques , Inhibiteurs de protéines kinases/pharmacologie , Reproductibilité des résultatsRÉSUMÉ
Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.
Sujet(s)
Leucine-rich repeat serine-threonine protein kinase-2/composition chimique , Modèles moléculaires , Domaines protéiques , Multimérisation de protéines , Séquence d'acides aminés , Domaine catalytique , Cristallographie aux rayons X , Cellules HEK293 , Humains , Leucine-rich repeat serine-threonine protein kinase-2/génétique , Leucine-rich repeat serine-threonine protein kinase-2/métabolisme , Mutation , Maladie de Parkinson/génétique , Maladie de Parkinson/métabolisme , Similitude de séquences d'acides aminésRÉSUMÉ
High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.
Sujet(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/métabolisme , Cytotoxines/pharmacologie , Tumeurs/thérapie , Pyridazines/composition chimique , Pyridazines/pharmacologie , Lignée cellulaire , Lignée cellulaire tumorale , Survie cellulaire/effets des médicaments et des substances chimiques , Cytotoxines/composition chimique , Cytotoxines/isolement et purification , Systèmes de délivrance de médicaments , Activation enzymatique/effets des médicaments et des substances chimiques , Antienzymes/pharmacologie , Génomique , Humains , ImmunotransfertRÉSUMÉ
The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11. Proteins 2016; 84(Suppl 1):34-50. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
Sujet(s)
Biologie informatique/statistiques et données numériques , Modèles moléculaires , Modèles statistiques , Protéines/composition chimique , Logiciel , Bactéries/composition chimique , Biologie informatique/méthodes , Infographie , Cristallographie aux rayons X , Bases de données de protéines , Humains , Coopération internationale , Pliage des protéines , Motifs et domaines d'intéraction protéique , Multimérisation de protéines , Structure secondaire des protéines , Similitude de séquences d'acides aminés , Virus/composition chimiqueRÉSUMÉ
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Sujet(s)
Bactériophages/génétique , Escherichia coli/virologie , Génomique/méthodes , Protéines virales/génétique , Escherichia coli/génétique , Escherichia coli/métabolisme , Génome viral , Protéines virales/biosynthèseRÉSUMÉ
Discerning the structural building blocks of macromolecules is essential for understanding their folding and function. For a new generation of modified nucleic acid ligands (called slow off-rate modified aptamers or SOMAmers), we previously observed essential functions of hydrophobic aromatic side chains in the context of well-known nucleic acid motifs. Here we report a 2.45-Å resolution crystal structure of a SOMAmer complexed with nerve growth factor that lacks any known nucleic acid motifs, instead adopting a configuration akin to a triangular prism. The SOMAmer utilizes extensive hydrophobic stacking interactions, non-canonical base pairing and irregular purine glycosidic bond angles to adopt a completely non-helical, compact S-shaped structure. Aromatic side chains contribute to folding by creating an unprecedented intercalating zipper-like motif and a prominent hydrophobic core. The structure provides compelling rationale for potent inhibitory activity of the SOMAmer and adds entirely novel motifs to the repertoire of structural elements uniquely available to SOMAmers.
Sujet(s)
ADN/composition chimique , Facteur de croissance nerveuse/composition chimique , Séquence d'acides aminés , Séquence nucléotidique , Sites de fixation , Cristallographie aux rayons X , Humains , Interactions hydrophobes et hydrophiles , Données de séquences moléculaires , Facteur de croissance nerveuse/physiologie , Liaison aux protéines , Structure secondaire des protéines , Technique SELEXRÉSUMÉ
Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.
Sujet(s)
Protéines de liaison au maltose/composition chimique , Protéine Mcl-1/composition chimique , Apoprotéines/composition chimique , Apoprotéines/génétique , Cristallisation , Cristallographie aux rayons X , Conception de médicament , Humains , Ligands , Protéines de liaison au maltose/génétique , Modèles moléculaires , Protéine Mcl-1/antagonistes et inhibiteurs , Protéine Mcl-1/génétique , Fragments peptidiques/composition chimique , Fragments peptidiques/génétique , Liaison aux protéines , Conformation des protéines , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/génétiqueRÉSUMÉ
Between 20% and 25% of patients diagnosed with Alzheimer's disease (AD) do not have amyloid burden as assessed by positron emission tomography imaging. Thus, there is a need for nonamyloid-directed therapies for AD, especially for those patients with non-amyloid AD. The family of phosphodiesterase-4 (PDE4) enzymes are underexploited therapeutic targets for central nervous system indications. While the PDE4A, B, and D subtypes are expressed in brain, the strict amino acid sequence conservation of the active site across the four subtypes of PDE4 has made it difficult to discover subtype inhibitors. The recent elucidation of the structure of the PDE4 N- and C-terminal regulatory domains now makes it possible to design subtype-selective, negative allosteric modulators (PDE4-NAMs). These act through closing the N-terminal UCR2 or C-terminal CR3 regulatory domains, and thereby inhibit the enzyme by blocking access of cyclic adenosine monophosphate (cAMP) to the active site. PDE4B-NAMs have the potential to reduce neuroinflammation by dampening microglia cytokine production triggered by brain amyloid, while PDE4D-NAMs have potent cognitive benefit by augmenting signaling through the cAMP/protein kinase A/cAMP response element-binding protein (CREB) pathway for memory consolidation. The importance of PDE4D for human cognition is underscored by the recent discovery of PDE4D mutations in acrodysostosis (ACRDY2: MIM 600129), an ultra rare disorder associated with intellectual disability. Thus, the family of PDE4 enzymes provides rich opportunities for the development of mechanistically novel drugs to treat neuroinflammation or the cognitive deficits in AD.
Sujet(s)
Maladie d'Alzheimer/enzymologie , Cyclic Nucleotide Phosphodiesterases, Type 4/métabolisme , Maladie d'Alzheimer/anatomopathologie , Animaux , HumainsRÉSUMÉ
In this study we report a series of triazine derivatives that are potent inhibitors of PDE4B. We also provide a series of structure activity relationships that demonstrate the triazine core can be used to generate subtype selective inhibitors of PDE4B versus PDE4D. A high resolution co-crystal structure shows that the inhibitors interact with a C-terminal regulatory helix (CR3) locking the enzyme in an inactive 'closed' conformation. The results show that the compounds interact with both catalytic domain and CR3 residues. This provides the first structure-based approach to engineer PDE4B-selective inhibitors.
Sujet(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/métabolisme , Découverte de médicament , Inhibiteurs de la phosphodiestérase/pharmacologie , Triazines/pharmacologie , Relation dose-effet des médicaments , Humains , Structure moléculaire , Inhibiteurs de la phosphodiestérase/synthèse chimique , Inhibiteurs de la phosphodiestérase/composition chimique , Relation structure-activité , Triazines/synthèse chimique , Triazines/composition chimiqueRÉSUMÉ
Phosphodiesterase-4B (PDE4B) regulates the pro-inflammatory Toll Receptor -Tumor Necrosis Factor α (TNFα) pathway in monocytes, macrophages and microglial cells. As such, it is an important, although under-exploited molecular target for anti-inflammatory drugs. This is due in part to the difficulty of developing selective PDE4B inhibitors as the amino acid sequence of the PDE4 active site is identical in all PDE4 subtypes (PDE4A-D). We show that highly selective PDE4B inhibitors can be designed by exploiting sequence differences outside the active site. Specifically, PDE4B selectivity can be achieved by capture of a C-terminal regulatory helix, now termed CR3 (Control Region 3), across the active site in a conformation that closes access by cAMP. PDE4B selectivity is driven by a single amino acid polymorphism in CR3 (Leu674 in PDE4B1 versus Gln594 in PDE4D). The reciprocal mutations in PDE4B and PDE4D cause a 70-80 fold shift in selectivity. Our structural studies show that CR3 is flexible and can adopt multiple orientations and multiple registries in the closed conformation. The new co-crystal structure with bound ligand provides a guide map for the design of PDE4B selective anti-inflammatory drugs.
Sujet(s)
Anti-inflammatoires/composition chimique , Anti-inflammatoires/synthèse chimique , Domaine catalytique/génétique , Inhibiteurs de la phosphodiestérase-4/composition chimique , Inhibiteurs de la phosphodiestérase-4/synthèse chimique , Séquence d'acides aminés/génétique , Anti-inflammatoires/pharmacologie , Domaine catalytique/effets des médicaments et des substances chimiques , Cristallographie aux rayons X , Cyclic Nucleotide Phosphodiesterases, Type 4/effets des médicaments et des substances chimiques , Conception de médicament , Macrophages/métabolisme , Microglie/métabolisme , Modèles moléculaires , Monocytes/métabolisme , Inhibiteurs de la phosphodiestérase-4/pharmacologie , Polymorphisme génétique , Facteur de nécrose tumorale alpha/biosynthèseRÉSUMÉ
For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins.
Sujet(s)
Biologie informatique/méthodes , Conformation des protéines , Protéines/composition chimique , Séquence d'acides aminés , Modèles moléculaires , Données de séquences moléculaires , Protéines/génétique , Alignement de séquencesRÉSUMÉ
Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.
Sujet(s)
Amidohydrolases/composition chimique , Bactériophages/enzymologie , Bactériophages/génétique , Synechococcus/virologie , Amidohydrolases/génétique , Amidohydrolases/métabolisme , Bactériophages/classification , Cristallographie aux rayons X , Modèles moléculaires , Photosynthèse , Complexe protéique du photosystème II/métabolisme , Phylogenèse , Spécificité du substrat , Synechococcus/physiologieRÉSUMÉ
Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.
Sujet(s)
Aptamères nucléotidiques/composition chimique , Aptamères nucléotidiques/métabolisme , Interactions hydrophobes et hydrophiles , Modèles moléculaires , Protéines proto-oncogènes c-sis/métabolisme , Technique SELEX/méthodes , Motifs d'acides aminés/génétique , Bécaplermine , Cristallographie aux rayons X , Amorces ADN/génétique , Données de séquences moléculaires , Structure moléculaire , Phosphorylation , Liaison aux protéines , Protéines proto-oncogènes c-sis/composition chimique , Analyse de séquence d'ADN , Température de transitionRÉSUMÉ
Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is available but in vitro propagation of the phage may not be practical or possible.