Arnaud Blondel

Tyrosine 319 in the Interdomain B of ZAP-70 Is a Binding Site for the Src Homology 2 Domain of Lck

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.

Drug discovery targeting amino acid racemases.

Drug Discovery Targeting Amino Acid Racemases Paola Conti, Lucia Tamborini, Andrea Pinto, Arnaud Blondel, Paola Minoprio, Andrea Mozzarelli, and Carlo De Micheli* Dipartimento di Scienze Farmaceutiche “P. Pratesi”, via Mangiagalli 25, 20133 Milano, Italy Institut Pasteur, Unit e de Bioinformatique Structurale, CNRS-URA 2185, D epartement de Biologie Structurale et Chimie, 25 rue du Dr. Roux, 75724 Paris, France Institut Pasteur, Laboratoire des Processus Infectieux a Trypanosoma; D epartement d’Infection et Epid emiologie; 25 rue du Dr. Roux, 75724 Paris, France Dipartimento di Biochimica e Biologia Molecolare, via G. P. Usberti 23/A, 43100 Parma, Italy Istituto di Biostrutture e Biosistemi, viale Medaglie d’oro, Roma, Italy

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 μM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.

Ensemble variance in free energy calculations by thermodynamic integration: Theory, optimal “Alchemical” path, and practical solutions

Thermodynamic integration is a widely used method to calculate and analyze the effect of a chemical modification on the free energy of a chemical or biochemical process, for example, the impact of an amino acid substitution on protein association. Numerical fluctuations can introduce large uncertainties, limiting the domain of application of the method. The parametric energy function describing the chemical modification in the thermodynamic integration, the “Alchemical path,” determines the amplitudes of the fluctuations. In the present work, I propose a measure of the fluctuations in the thermodynamic integration and an approach to search for a parametric energy path minimizing that measure. The optimal path derived with this approach is very close to the theoretical minimum of the measure, but produces nonergodic sampling. Nevertheless, this path is used to guide the design of a practical and efficient path producing correct sampling. The convergence with this practical path is evaluated on test cases, and compares favorably with that of other methods such as power or polynomial path, soft‐core van der Waals, and some other approaches presented in the literature.

The conformational plasticity of calmodulin upon calcium complexation gives a model of its interaction with the oedema factor of Bacillus anthracis

We analyzed the conformational plasticity of calmodulin (CaM) when it is bound to the oedema factor (EF) of Bacillus anthracis and its response to calcium complexation with molecular dynamics (MD) simulations. The EF‐CaM complex was simulated during 15 ns for three different levels of calcium bound to CaM. They were respectively no calcium ion (EF–(Apo‐CaM)), two calcium ions bound to the C‐terminal domain of CaM (EF–(2Ca‐CaM)), and four calcium ions bound to CaM (EF–(4Ca‐CaM)). Calculations were performed using AMBER package. The analysis of the MD simulations illustrates how CaM forces EF in an open conformation to form the adenylyl cyclase enzymatic site, especially with the two calcium form of CaM, best suited to fit the open conformation of EF. By contrast, CaM encounters bending and unwinding of its flexible interlinker in EF–(Apo‐CaM) and EF–(4Ca‐CaM). Calcium binding to one domain of CaM affects the other one, showing a transmission of information along the protein structure. The analysis of the CaM domains conformation along the simulations brings an atomistic and dynamic explanation for the instability of these complexes. Indeed the EF‐hand helices of the N‐terminal domain tend to open upon calcium binding (EF–(4Ca‐CaM)), although the domain is locked by EF. By contrast, the C‐terminal domain is strongly locked in the open conformation by EF, and the removal of calcium induces a collapse of EF catalytic site (EF–(Apo‐CaM)). Proteins 2008.

Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins

Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities.

A fast and convenient way to produce single stranded DNA from a phagemid

Despite the increased number of methods using double stranded DNA for sequencing, site directed mutagenesis, e t c . , it is often more convenient to use vectors able to produce single stranded DNA for such usages. The phagemids are more convenient for many purposes in molecular biology than M13 phage because they are smaller and can grow on bacteria as colonies instead of plaques, However the obtention of single stranded DNA from phagemids is longer than from M13 since it needs an infection step with a helper phage. A convenient protocol for single stranded DNA production from phagemids is proposed here. Used with pEMBL8 and derived vectors, this method provides similar yields than other described methods (1-3). It avoids absorbance monitored culture, saves up to one day and is far less consuming in helper phage stock. A colony of male E. coli strain harboring the phagemid of interest, taken from a fresh selective medium plate (less than one week-old), is suspended in 10-50 /xl of 2YT medium. The bacterial suspension is then infected by addition of 1 y\ of M13 KO7 helper phage stock (more than 10 pfu/ml) and incubated 15 min at RT. 500 /tl of 2YT with antibiotic for phagemid selection are added to the infected cells which are then incubated at 37 °C for one hour to let helper phage express antibiotic resistance. 100 to 200 /tl of incubated cells are added to 3—4 ml of 2YT medium with appropriate antibiotics (we used 150 mg/1 ampicillin and 75 mg/1 kanamycin), The culture is proceeded for 18-20 hours at 37°C. The culture should be lead to saturation for good single strand production. The virion particles are isolated and single stranded DNA is prepared as in (3). 1.5 ml culture leading to 30 /tl of preparation should provide concentrations suitable for most applications (see figure).

Impact of M36I polymorphism on the interaction of HIV-1 protease with its substrates: insights from molecular dynamics

BackgroundOver the last decades, a vast structural knowledge has been gathered on the HIV-1 protease (PR). Noticeably, most of the studies focused the B-subtype, which has the highest prevalence in developed countries. Accordingly, currently available anti-HIV drugs target this subtype, with considerable benefits for the corresponding patients.However, in developing countries, there is a wide variety of HIV-1 subtypes carrying PR polymorphisms related to reduced drug susceptibility. The non-active site mutation, M36I, is the most frequent polymorphism, and is considered as a non-B subtype marker.Yet, the structural impact of this substitution on the PR structure and on the interaction with natural substrates remains poorly documented.ResultsHerein, we used molecular dynamics simulations to investigate the role of this polymorphism on the interaction of PR with six of its natural cleavage-sites substrates.Free energy analyses by MMPB/SA calculations showed an affinity decrease of M36I-PR for the majority of its substrates. The only exceptions were the RT-RH, with equivalent affinity, and the RH-IN, for which an increased affinity was found. Furthermore, molecular simulations suggest that, unlike other peptides, RH-IN induced larger structural fluctuations in the wild-type enzyme than in the M36I variant.ConclusionsWith multiple approaches and analyses we identified structural and dynamical determinants associated with the changes found in the binding affinity of the M36I variant. This mutation influences the flexibility of both PR and its complexed substrate. The observed impact of M36I, suggest that combination with other non-B subtype polymorphisms, could lead to major effects on the interaction with the 12 known cleavage sites, which should impact the virion maturation.

Molecular Motions as a Drug Target: Mechanistic Simulations of Anthrax Toxin Edema Factor Function Led to the Discovery of Novel Allosteric Inhibitors

Edema Factor (EF) is a component of Bacillus anthracis toxin essential for virulence. Its adenylyl cyclase activity is induced by complexation with the ubiquitous eukaryotic cellular protein, calmodulin (CaM). EF and its complexes with CaM, nucleotides and/or ions, have been extensively characterized by X-ray crystallography. Those structural data allowed molecular simulations analysis of various aspects of EF action mechanism, including the delineation of EF and CaM domains through their association energetics, the impact of calcium binding on CaM, and the role of catalytic site ions. Furthermore, a transition path connecting the free inactive form to the CaM-complexed active form of EF was built to model the activation mechanism in an attempt to define an inhibition strategy. The cavities at the surface of EF were determined for each path intermediate to identify potential sites where the binding of a ligand could block activation. A non-catalytic cavity (allosteric) was found to shrink rapidly at early stages of the path and was chosen to perform virtual screening. Amongst 18 compounds selected in silico and tested in an enzymatic assay, 6 thiophen ureidoacid derivatives formed a new family of EF allosteric inhibitors with IC50 as low as 2 micromolars.

Discrimination of agonists versus antagonists of nicotinic ligands based on docking onto AChBP structures.

Numerous high-resolution crystallographic structures of the acetylcholine binding protein (AChBP), a molluscan cholinergic protein, homologous to the extracellular domain of nicotinic acetylcholine receptors, are available. This offers opportunities to model the interaction between various ligands and the acetylcholine binding site. Herein we present a study of the interplay between ligand binding and motions of the C-loop capping the binding site. Nicotinic agonists and antagonists were docked on AChBP X-ray structures. It is shown that the studied agonists and antagonists can be discriminated according to their higher affinities for structures respectively obtained in the presence of agonists or antagonists, highlighting the fact that AChBP structures retain a pharmacological footprint of the compound used in crystallography experiments. A detailed analysis of the binding site cavities suggests that this property is mainly related to the shape of the cavities.