Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

18 pags., 6 figs., 1 tab. -- Open Access funded by Creative Commons Atribution Licence 4.0Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from INSTRUCT (“Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301“), FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank the ESRF for beamtime at ID30A and ID23-1. Funding: This work was supported by Spanish Ministerio de Economia y Competitividad (BFU2016-78232-P) and Instituto de Salud Carlos III co-funded by European Union (PI15/00663 and PI18/00349, ERDF/ ESF, “Investing in your future”). This work was financially supported by the European Research Council (ERC-Stg-2012-311318 to P.S.). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017)

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design

Bioinformatics and molecular modeling in glycobiology

The field of glycobiology is concerned with the study of the structure, properties, and biological functions of the family of biomolecules called carbohydrates. Bioinformatics for glycobiology is a particularly challenging field, because carbohydrates exhibit a high structural diversity and their chains are often branched. Significant improvements in experimental analytical methods over recent years have led to a tremendous increase in the amount of carbohydrate structure data generated. Consequently, the availability of databases and tools to store, retrieve and analyze these data in an efficient way is of fundamental importance to progress in glycobiology. In this review, the various graphical representations and sequence formats of carbohydrates are introduced, and an overview of newly developed databases, the latest developments in sequence alignment and data mining, and tools to support experimental glycan analysis are presented. Finally, the field of structural glycoinformatics and molecular modeling of carbohydrates, glycoproteins, and protein–carbohydrate interaction are reviewed

Testing a global standard for quantifying species recovery and assessing conservation impact

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a “Green List of Species” (now the IUCN Green Status of Species). A draft Green Status framework for assessing species’ progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species’ viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species’ recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard

Listeria pathogenesis and molecular virulence determinants

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research

Determinants of Aedes mosquito density as an indicator of arbovirus transmission risk in three sites affected by co-circulation of globally spreading arboviruses in Colombia, Ecuador and Argentina

Background: The global impact of Zika virus in Latin America has drawn renewed attention to circulating mosquito-borne viruses in this region, such as dengue and chikungunya. Our objective was to assess socio-ecological factors associated with Aedes mosquito vector density as a measure of arbovirus transmission risk in three cities of potentially recent Zika virus introduction: Ibagué, Colombia; Manta, Ecuador; and Posadas, Argentina, in order to inform disease mitigation strategies. Methods: We sampled Aedes mosquito populations in a total of 1086 households, using indoor and peridomestic mosquito collection methods, including light traps, resting traps, traps equipped with chemical attractant and aspirators. For each sampled household, we collected socio-economic data using structured questionnaires and data on microenvironmental conditions using iButton data loggers. Results: A total of 3230 female Aedes mosquitoes were collected, of which 99.8% were Aedes aegypti and 0.2% were Aedes albopictus. Mean female Aedes mosquito density per household was 1.71 (standard deviation: 2.84). We used mixed-effects generalized linear Poisson regression analyses to identify predictors of Aedes density, using month, neighborhood and country as random-effects variables. Across study sites, the number of household occupants [incidence rate ratio (IRR): 1.08, 95% confidence interval (CI): 1.01–1.14], presence of entry points for mosquitoes into the household (IRR: 1.51, 95% CI: 1.30–1.76) and presence of decorative vegetation (IRR: 1.52, 95% CI: 1.22–1.88) were associated with higher Aedes density; while being in the highest wealth tertile of household wealth (IRR: 0.78, 95% CI: 0.66–0.92), knowledge of how arboviruses are transmitted (IRR: 0.94, 95% CI: 0.89–1.00) and regular emptying of water containers by occupants (IRR: 0.79, 95% CI: 0.67–0.92) were associated with lower Aedes density. Conclusions: Our study addresses the complexities of arbovirus vectors of global significance at the interface between human and mosquito populations. Our results point to several predictors of Aedes mosquito vector density in countries with co-circulation of multiple Aedes-borne viruses, and point to modifiable risk factors that may be useful for disease prevention and control.Fil: Talbot, Benoit. University of Ottawa; CanadáFil: Sander, Beate. University of Toronto; CanadáFil: Cevallos, Varsovia. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: González, Camila. Universidad de los Andes; ColombiaFil: Benítez, Denisse. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: Carissimo, Claudio. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Carrasquilla Ferro, María C.. Universidad de los Andes; ColombiaFil: Gauto, Neris. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Litwiñiuk, Sergio Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; ArgentinaFil: López, Karen. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Ortiz, Mario I.. Universidad de los Andes; ColombiaFil: Ponce, Patricio. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: Villota, Stephany D.. Ministerio de Salud Pública. Instituto Nacional de Investigación en Salud Pública Dr. Leopoldo Izquieta Perez; EcuadorFil: Zelaya, Fabian. Provincia de Misiones. Municipalidad de Posadas; ArgentinaFil: Espinel, Mauricio. Universidad Tecnica de Manabi; EcuadorFil: Wu, Jianhong. University of York; Reino UnidoFil: Miretti, Marcos Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; ArgentinaFil: Kulkarni, Manisha A.. University of Ottawa; Canad

Testing a global standard for quantifying species recovery and assessing conservation impact.

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a "Green List of Species" (now the IUCN Green Status of Species). A draft Green Status framework for assessing species' progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species' viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species' recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard