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SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

International audience; Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

Summary Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO – haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role.

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring. DOI: http://dx.doi.org/10.7554/eLife.21422.001

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John WileySons, Ltd.

International audience; The " 7 kDa DNA-binding " family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative " 7 kDa DNA-binding " family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents. In living organisms, the long genomic DNA has to be packed in order to fit into cells, while the genetic information must stay accessible for replication and transcription events. To this aim, organisms have developed different compaction systems, such as the wrapping of DNA around histones to form the chromatin in Eukarya, and the supercoiling of DNA with the help of non-histone proteins to form the nucleoid in Bacteria. Archaea often live in extreme environments and have the additional challenge to protect their genomic DNA from extreme conditions, such as high temperatures. Many Archaea contain homologs of eukaryotic histones, but Desulfurococcales, Thermoplasmatales and Sulfolobales use a different kind of packaging proteins 1,2. Hyperthermophile and acidophile archaea of the Sulfolobales order from the Crenarchaeota kingdom express small basic DNA-binding proteins, which represent about 5% of the total soluble cellular proteins, sufficient to coat the entire genome of a Sulfolobus cell 3. These proteins constitute the family called " 7 kDa DNA-binding " or Sul7d 4. They were first isolated from Sulfolobus acido-caldarius which produces five of them, named Sac7a, b, c, d, and e. Sac7d and Sac7e are encoded by distinct genes, while Sac7a and b are truncated versions of Sac7d 5–7. Highly similar homologs have been found in all Sulfolobus species, such as Sso7d from Sulfolobus solfataricus 8 , and Ssh7a and Ssh7b from Sulfolobus shibatae-two proteins encoded by two distinct genes 3,9. Sac7d and Sso7d are the two most studied proteins of this family. They have been characterized for their structure, function, chemical stability and biophysical properties 7. Sac7d and Sso7d are hyperthermostable (T m = 90.4 °C and 100.2 °C, respectively) 10,11 and are resistant from pH 0 up to at least pH 12 12,13. Although Sac7d and Sso7d sequences show only few differences, Sso7d is more stable than Sac7d. Their three-dimensional structures show that they both fold as an SH3-like domain capped by a C-terminal α-helix 14,15 and that they sharply kink the double DNA helix upon binding into the minor groove 16,17. It has been shown that Sac7d and Sso7d are general dsDNA binders with K D values varying in a salt dependent manner from 20 nM (low salt) to 3.8 μ M (high salt) for Sac7d, and from 116 nM to 12.8 μ M for Sso7d, and with a preference for G/C rich sequences 18,19. Sac7d has the property to increase the thermal stability of DNA duplexes by as much as 43.5 °C 6,15. Furthermore, Ssh7a and Ssh7b have been partially characterized and an affinity for dsDNA of about 100 nM

International audience; Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.

The capability of adsorption of different electroactive cationic Re(V)–amine complexes onto myoglobin-containing electrodes has been investigated. The goal of this work was the development of an Au/thiol/myo electrode and, after incubation of such ensemble in the presence of three different Re(V)–amine complexes, the evaluation of the extent of surface coverage by the complexes (as a way to evaluate the interaction complex-protein) using electrochemical techniques. Our results showed that a protein-containing electrode could therefore be used for the detection of the interaction of small electroactive cationic complexes and the biomolecule. The extent of the coverage of the myoglobin electrode by the complex depends on the number of free tails from the ligands and the total charge of the complex.

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

Retroviral capsids in their native form Capsid proteins of retroviruses form protective lattices around viral RNA molecules. The precise molecular details of how individual, full-length capsid proteins assemble to shield the viral genome; however, are not well understood. Obal et al. and Gres et al. now report high resolution crystal structures of the full length capsid proteins from Bovine Leukemia Virus and HIV-1, respectively. The two studies complement each other to reveal the dynamic nature of capsid protein assembly and of how individual capsid proteins interact in the lattice. The findings may have relevance for drug design. Science , this issue p. 95 ; see also p. 99

A reliable method based on electrochemical measurements for the evaluation of protein interaction with small electroactive coordination compounds is proposed. Protein binding capacity of five different cationic Re(V) complexes was evaluated by means of voltammetric techniques in the absence and the presence of bovine serum albumin. The percentage of interaction between the complex and the protein was estimated from the quantitative decrease of the diffusion coefficient for the sole complex due to the addition of the protein, and therefore the formation of the protein-complex molecular ensemble. The proposed methodology was checked using cisplatin as a molecular complex probe.

Recombinant protein expression has become an invaluable tool for academic and biotechnological projects. With the use of high-throughput screening technologies for soluble protein production, uncountable target proteins were produced in a soluble and homogeneous state enabling the realization of further studies. Evaluation of hundreds conditions requires the use of high-throughput cloning and screening methods. Here we describe a new versatile vector suite dedicated to the expression improvement of recombinant proteins with solubility problems. This vector suite allows the parallel cloning of the same PCR product into the 12 different expression vectors evaluating protein expression under different promoter strength, different fusion tags as well as different solubility enhancer proteins. Additionally, we propose the use of a new fusion protein which appear to be a useful solubility enhancer. Overall in this work we propose an economic and useful vector suite to fast track the solubility of different recombinant proteins and we propose a new solubility enhancer protein that can be included in the evaluation of the expression of recombinant proteins that are insoluble in classical expression conditions.

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.

International audience; The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.

International audience; Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

Mitogen-activated protein kinases (MAPKs) are involved in environmental signal sensing. They are thus expected to play key roles in the biology of Trypanosomatid parasites, which display complex life cycles and use extracellular cues to modulate cell differentiation. Despite their relevance, structural data of Trypanosomatid MAPKs is lacking. We have now determined the crystal structure of Leishmania major LmaMPK10, a stage-specifically activated MAPK, both alone and in complex with SB203580. LmaMPK10 was observed to be more similar to p38 than to other human MAPKs. However, significant differences could be identified in the catalytic pocket, as well as in potentially regulatory sites in the N-terminal lobe. The modified pocket architecture in LmaMPK10 precludes DFG-in/DFG-out regulatory flipping as observed in mammalian MAPKs. LmaMPK10-nucleotide association was also studied, revealing a potential C-terminal autoinhibitory mechanism. Overall, these data should speed the discovery of molecules interfering with LmaMPK10 functions, with relevance for antileishmanial drug development strategies. Fil: Horjales, Sofía. Instituto Pasteur de Montevideo; Uruguay Fil: Schmidt Arras, Dirk. Instituto Pasteur; Francia Fil: Rodriguez Limardo, Ramiro Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Leclercq, Olivier. Instituto Pasteur; Francia Fil: Obal, Gonzalo. Instituto Pasteur de Montevideo; Uruguay Fil: Prina, Eric. Instituto Pasteur; Francia Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Späth, Gerald F.. Instituto Pasteur; Francia Fil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; Uruguay. Instituto Pasteur; Francia

In Uruguay, more than 50% of dairy cattle individuals are infected by Bovine Leukemia Virus (BLV). A main goal of our country is to decrease this extremely high prevalence by developing efficient eradication programs for this disease. The aim of this study was to develop a rapid and sensitive real time PCR assay using SYBR green chemistry to detect and quantify BLV proviral DNA by amplifying gp51 gene from bovine peripheral blood. By using plasmid containing gp51 gene diluted in non-infected bovine genomic DNA we could determine the assay sensitivity. A comparative analysis with validated diagnostic tests (AGID, ELISA and direct nested PCR) was performed in 45 dairy cattle samples. All AGID positive animals (n=14) were positive by ELISA, while three negative AGID samples were also positive by ELISA. All ELISA positive animals (n=17) were positive by nested PCR. Real-time PCR technique shown that 15 out of 17 positive ELISA samples were positive whereas 10 out of 28 negative ELISA samples were also positive. These results reveal high agreement with nested PCR, and confirm an increased sensitivity of the PCR (real-time and nested) over the ELISA and AGID tests respectively. Overall, our results show that this SYBR Green -based PCR assay may be a useful, simple, and rapid tool to detect BLV infection in dairy cattle samples that could be adapted to high-troughput diagnostic procedures.

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the correct conformation of the produced protein is crucial. There is no single method to assess every aspect of protein structure or function. Depending on the protein, the methods of choice vary. There are general methods to evaluate not only mass and primary sequence of the protein, but also higher-order structure. This review outlines the principal techniques for determining the conformation of a protein from structural (biophysical methods) to functional (in vitro binding assays) analyses.

International audience; Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite-host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be β-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thrphosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His6-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes. Agencia Nacional de Investigación e Innovación

International audience; Bovine leukaemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus of the family Retroviridae. Recent studies revealed that BLV strains can be classified into six different genotypes and raised the possibility that another genotype may exist. In order to gain insight into the degree of genetic variability of BLV strains circulating in the South American region, a phylogenetic analysis was performed using gp51 env gene sequences. The results of these studies revealed the presence of seven BLV genotypes in this geographic region and the suitability of partial gp51 env gene sequences for phylogenetic inference. A significant number of amino acid substitutions found in BLV strains isolated in South America map to the second neutralization domain of gp51. A 3D molecular model of BLV gp51 revealed that these substitutions are located on the surface of the molecule. This may provide a selective advantage to overcome immune host neutralization.

International audience; To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. (C) 2008 Elsevier Inc. All rights reserved.

myo-Inositol hexakisphosphate (InsP6) is an ubiquitous and abundant molecule in the cytosol and nucleus of eukaryotic cells whose biological functions are incompletely known. A major hurdle for studying the biology of InsP6 has been a deficiency of a full understanding of the chemistry of its interaction with divalent and trivalent cations. This deficiency has limited our appreciation of how it remains in solution within cells, and the likely degree to which it might interact in vivo with physiologically important cations such as Ca2+ and Fe3+. We report here the initial part of the description of the InsP6-multivalent cation chemistry, including its solution equilibria studied by high resolution potentiometry and (for the Fe(III)/Fe(II) couple) cyclic voltammetry. InsP6 forms anionic complexes of high affinities and 1:1 stoichiometry with Mg(II), Ca(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II). Of particular importance is the observation that, in the exceptional case of Mg(II), InsP6 forms the species [Mg5(H2L)] (L representing fully deprotonated InsP6); this soluble neutral species is predicted to be the predominant form of InsP6 under nuclear or cytosolic conditions in animal cells. Contrary to previous suggestions, InsP6 is predicted not to interact with cytosolic calcium even when calcium is increased during signalling events. In vitro, InsP6 also forms high affinity 1:1 complexes with Fe(III) and Al(III). However, our data predict that in the biological context of excess free Mg(II), neither Fe(III) nor Fe(II) are complexed by InsP6.

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

The electrochemical behavior of trans-[Re((V))O(2)(en)(2)]I and trans-[Re((V))O(2) (en)(2)]ClO(4) (en=ethylenediamine) complexes was studied by cyclic voltammetry on Au electrodes. Experiments were performed in aqueous solutions at pH 7.0 and at room temperature. The complex voltammogram was characterized by Re-containing species, assigned to the [Re((V))O(2)(en)(2)](+)/[Re((IV))O(2)(en)(2)] couple, and I-containing species. To overcome I interference, the electrochemical response of Re complexes was segregated by performing a reductive desorption of adsorbed I from Au. Copyright 2001 Academic Press.

Bovine leukemia virus (BLV) and Human T-cell Leukemia virus (HTLV) are oncogenic retroviruses of the genus Deltaretrovirus, and affects cattle and human respectively. BLV is the etiologic agent of Enzootic Bovine Leukemia and infects B-cells of dairy/beef cattle generating a life-long infection leading to economic losses and commercial restraints. In common with other retroviruses, formation of a functional core structure during morphogenesis of BLV/HTLV viral particles is essential for infectivity. In this process, thousands of capsid (CA) molecules self-assemble to form a shell which encases the genome. The assembly mechanism of BLV-CA protein assemble to form mature-type core in vivo and in vitro are unknown, and more broadly, they are yet poorly understood in Deltaretrovirus, despite important advances utilizing HTLV-CA protein. We recently started in vitro analysis of the characteristics/requirements of BLV-CA protein assembly and developed a turbidimetry-based assembly assay using the purified recombinant BLV-CA protein. Here we show the examination of assembly under a variety of environmental and physical, including near physiological, conditions. Specifically, the effect on self-assembly triggering and kinetics was analysed for protein concentration, pH, ionic strength, temperature, phosphate and polyphosphates. The influence of both independent N-terminal and C-terminal domains on oligomerization kinetics was also assessed. In parallel, we performed electron microscopic analysis of assembly products in order to evaluate the physical characteristics of the material formed under these conditions. This work provides the first description of the BLV capsid protein assembly properties, which may also be of relevance for understanding other Deltaretrovirus assembly characteristics.