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Vincentelli R.,CNRS Architecture and Functions of Biological Macromolecules Lab | Romier C.,University of Strasbourg
Current Opinion in Structural Biology | Year: 2013

Escherichia coli is the major expression host for the production of homogeneous protein samples for structural studies. The introduction of high-throughput technologies in the last decade has further revitalized E. coli expression, with rapid assessment of different expression strategies and successful production of an ever-increasing number of proteins. In addition, miniaturization of biophysical characterizations should soon help choosing expression strategies based on quantitative and qualitative observations. Since many proteins form larger assemblies in vivo, dedicated co-expression systems for E. coli are now addressing the reconstitution of protein complexes. Yet, co-expression approaches show an increasing experimental combinatorial intricacy when considering larger complexes. The current combination of high-throughput and co-expression technologies paves the way, however, for tackling larger and more complex macromolecular assemblies. © 2013.


Desmyter A.,CNRS Architecture and Functions of Biological Macromolecules Lab | Spinelli S.,CNRS Architecture and Functions of Biological Macromolecules Lab | Roussel A.,CNRS Architecture and Functions of Biological Macromolecules Lab | Cambillau C.,CNRS Architecture and Functions of Biological Macromolecules Lab
Current Opinion in Structural Biology | Year: 2015

In recent years, the use of single-domain camelid immunoglobulins, termed vHHs or nanobodies, has seen increasing growth in biotechnology, pharmaceutical applications and structure/function research. The usefulness of nanobodies in structural biology is now firmly established, as they provide access to new epitopes in concave and hinge regions - and stabilize them. These sites are often associated with enzyme inhibition or receptor neutralization, and, at the same time, provide favorable surfaces for crystal packing. Remarkable results have been achieved by using nanobodies with flexible multi-domain proteins, large complexes and, last but not least, membrane proteins. While generating nanobodies is still a rather long and expensive procedure, the advent of naive libraries might be expected to facilitate the whole process. © 2015 Elsevier Ltd.


Hemsworth G.R.,University of York | Henrissat B.,CNRS Architecture and Functions of Biological Macromolecules Lab | Davies G.J.,University of York | Walton P.H.,University of York
Nature chemical biology | Year: 2014

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.


Habchi J.,CNRS Architecture and Functions of Biological Macromolecules Lab | Longhi S.,CNRS Architecture and Functions of Biological Macromolecules Lab
Molecular BioSystems | Year: 2012

This review focuses on the experimental data showing the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely Nipah (NiV), Hendra (HeV) and measles (MeV) viruses. We provide a detailed description of the molecular mechanisms governing the disorder-to-order transition of the intrinsically disordered C-terminal domains (N TAIL) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins. We also show that a significant flexibility persists within N TAIL-XD complexes, which therefore provide illustrative examples of "fuzziness". The functional implications of structural disorder are discussed in light of the ability of disordered regions to establish a complex molecular partnership, thereby leading to a variety of biological effects. Taking into account the promiscuity that typifies disordered regions, we propose that the main functional advantage of the abundance of disorder within viruses would reside in pleiotropy and genetic compaction, where a single gene would encode a single (regulatory) protein product able to establish multiple interactions via its disordered regions, and hence to exert multiple concomitant biological effects.


Durand E.,Aix - Marseille University | Durand E.,CNRS Architecture and Functions of Biological Macromolecules Lab | Cambillau C.,Aix - Marseille University | Cascales E.,CNRS Architecture and Functions of Biological Macromolecules Lab | Journet L.,CNRS Architecture and Functions of Biological Macromolecules Lab
Trends in Microbiology | Year: 2014

The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into both prokaryotic and eukaryotic cells, therefore participating in interbacterial competition and virulence. The T6SS is functionally and structurally similar to the contractile bacteriophage cell puncturing device: the contraction of a sheath-like structure is believed to propel an inner tube terminated by a spike towards target cells, allowing the delivery of effectors. In this review, we summarize recent advances in the identification and characterization of T6SS effector proteins, highlighting the broad repertoire of enzymatic activities, and discuss recent findings relating to the secretion mechanisms. © 2014 Elsevier Ltd.


Longhi S.,CNRS Architecture and Functions of Biological Macromolecules Lab
Advances in Experimental Medicine and Biology | Year: 2012

In this chapter, I focus on the biochemical and structural characterization of the complex between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N TAIL) and the C-terminal X domain (XD) of the viral phosphoprotein (P). I summarize the main experimental data available so far pointing out the prevalently disordered nature of N TAIL even after complex formation and the role of the flexible C-terminal appendage in the binding reaction. I finally discuss the possible functional role of these residual disordered regions within the complex in terms of their ability to capture other regulatory, binding partners. © 2012 Landes Bioscience and Springer Science+Business Media.


Cascales E.,Aix - Marseille University | Cambillau C.,CNRS Architecture and Functions of Biological Macromolecules Lab
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2012

Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of proteins or domains directly into target cells. These systems are versatile as they can target either eukaryotic host cells and therefore modulate the bacteria-host interaction and pathogenesis or bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise at least 13 proteins. Although recent years have witnessed advances in the role and function of these secretion systems, little is known about how these complexes assemble in the cell envelope. Interestingly, the current information converges to the idea that T6SSs are composed of two subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to the cell envelope. In this review, we summarize recent structural information on individual T6SS components emphasizing the fact that T6SSs are composite systems, adapting subunits from various origins. ©2012 The Royal Society.


Medie F.M.,Aix - Marseille University | Medie F.M.,CNRS Architecture and Functions of Biological Macromolecules Lab | Davies G.J.,University of York | Drancourt M.,Aix - Marseille University | Henrissat B.,CNRS Architecture and Functions of Biological Macromolecules Lab
Nature Reviews Microbiology | Year: 2012

Cellulolytic enzymes have been the subject of renewed interest owing to their potential role in the conversion of plant lignocellulose to sustainable biofuels. An analysis of ∼1,500 complete bacterial genomes, presented here, reveals that ∼40% of the genomes of sequenced bacteria encode at least one cellulase gene. Most of the bacteria that encode cellulases are soil and marine saprophytes, many of which encode a range of enzymes for cellulose hydrolysis and also for the breakdown of the other constituents of plant cell walls (hemicelluloses and pectins). Intriguingly, cellulases are present in organisms that are usually considered as non-saprophytic, such as Mycobacterium tuberculosis, Legionella pneumophila, Yersinia pestis and even Escherichia coli. We also discuss newly emerging roles of cellulases in such non-saprophytic organisms. © 2012 Macmillan Publishers Limited. All rights reserved.


Cantarel B.L.,University of Maryland, Baltimore | Lombard V.,CNRS Architecture and Functions of Biological Macromolecules Lab | Henrissat B.,CNRS Architecture and Functions of Biological Macromolecules Lab
PLoS ONE | Year: 2012

The various ecological habitats in the human body provide microbes a wide array of nutrient sources and survival challenges. Advances in technology such as DNA sequencing have allowed a deeper perspective into the molecular function of the human microbiota than has been achievable in the past. Here we aimed to examine the enzymes that cleave complex carbohydrates (CAZymes) in the human microbiome in order to determine (i) whether the CAZyme profiles of bacterial genomes are more similar within body sites or bacterial families and (ii) the sugar degradation and utilization capabilities of microbial communities inhabiting various human habitats. Upon examination of 493 bacterial references genomes from 12 human habitats, we found that sugar degradation capabilities of taxa are more similar to others in the same bacterial family than to those inhabiting the same habitat. Yet, the analysis of 520 metagenomic samples from five major body sites show that even when the community composition varies the CAZyme profiles are very similar within a body site, suggesting that the observed functional profile and microbial habitation have adapted to the local carbohydrate composition. When broad sugar utilization was compared within the five major body sites, the gastrointestinal track contained the highest potential for total sugar degradation, while dextran and peptidoglycan degradation were highest in oral and vaginal sites respectively. Our analysis suggests that the carbohydrate composition of each body site has a profound influence and probably constitutes one of the major driving forces that shapes the community composition and therefore the CAZyme profile of the local microbial communities, which in turn reflects the microbiome fitness to a body site. © 2012 Cantarel et al.


Habchi J.,Aix - Marseille University | Habchi J.,CNRS Architecture and Functions of Biological Macromolecules Lab | Tompa P.,Vrije Universiteit Brussel | Tompa P.,Hungarian Academy of Sciences | And 4 more authors.
Chemical Reviews | Year: 2014

Proteins are the major component of the living cell. They play crucial roles in the maintenance of life, and their dysfunctions are known to cause different pathologies. Simple amino acid propensities reflect some basic physical or sequence features. Such propensity-based predictors rely on simple statistics of amino acid propensity, on the physical/chemical features of amino acids, or on a preliminary concept on the physical background of disorder. Regions of missing electron density in the PDB are generally short, as long regions prevent crystallization. As such, short disorder is overrepresented in the database of disordered regions, and hence these predictors tend to perform better in predicting short disorder than long disorder. Predictors can also be classified based on the binary nature of the prediction. Examples of binary predictors are the CH plot and the cumulative distribution function (CDF) analysis.

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