J. Lescar - Academia.edu (original) (raw)

Papers by J. Lescar

The tRNA (m 1 G37) methyltransferase TrmD catalyzes m 1 G formation at position 37 in many tRNA i... more The tRNA (m 1 G37) methyltransferase TrmD catalyzes m 1 G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-L-methionine (SAM) and tRNA Leu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNA Leu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (K i = 0.41 ± 0.07 µM) and uncompetitive for tRNA (K i = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

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Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified... more Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents. IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

General physiology and biophysics, 1998

Structure, 2001

virus (HIV) are obtained by posttranslational cleavage of the Gag, Gag/Pol, and Env viral polypro... more virus (HIV) are obtained by posttranslational cleavage of the Gag, Gag/Pol, and Env viral polyprotein precur-Institute of Molecular Genetics Academy of Sciences of the Czech Republic sors. The Gag and Gag/Pol polyproteins, in particular, are cleaved by a viral protease, which is itself contained Flemingovo nam. 2 166 37 Prague 6 within the Gag/Pol polyprotein chain. Since the demonstration that the human immunodeficiency virus prote-Czech Republic 2 Centre de Recherches sur les Macromolé cules ase (HIV Pr) is essential in the viral life cycle [1-3], this enzyme has become one of the primary targets for anti-Vé gé tales (affiliated with Université Joseph Fourier)-CNRS BP53 viral drug design. This has led to the development of many active-site inhibitors, some of which are currently F38041 Grenoble Cedex France in use as therapeutic agents for AIDS treatment [4]. In order to be proteolytically active, two protease monomers 3 European Synchrotron Radiation Facility BP220 must assemble into a homodimer, with each subunit contributing residues to the substrate binding pocket, F38043 Grenoble Cedex France including residue Asp-25, which is directly involved in the catalysis [5]. The requirement for protease dimeriza-4 Unité d'Immunologie Structurale (URA 1961 CNRS) tion to achieve proteolytic activity has led several authors to propose alternative noncompetitive inhibitors Dé partement d'Immunologie Institut Pasteur that would provoke the dissociation of the active homodimeric assembly [6-8]. 75724 Paris Cedex 15 France With the objective of probing the structural stability of HIV Pr and the eventual design of potential inhibitors of the enzyme that are directed to regions other than the active site, we have examined the effects of anti-Summary HIV-1 Pr monoclonal antibodies (mAbs) on the catalytic activity of the protease [9, 10]. We have recently reported Background: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is a preliminary study of mAb1696, which, although raised against the HIV-1 Pr, inhibits the catalytic activity of both essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The the HIV-1 and HIV-2 enzymes with inhibition constants of 0.6 nM and 1.5 nM, respectively, at pH 7.4 [11]. This murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the study also showed that mAb1696 cross-reacts with peptides containing the N terminus of the HIV protease. The catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nano-N-terminal region accounts for a large percentage of the interface between the two HIV Pr monomers because it molar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that interdigitates with the C-terminal segment from the other monomer, thus forming a 4-stranded intermolecu-is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimer-lar ␤-pleated sheet in the active homodimer. We proposed that mAb1696 inhibits HIV Pr by perturbing the ization to the active enzymatic form. native structure of the enzyme at the dimer interface [11]. In addition, a clustering of negatively charged resi-Results: We report here the crystal structure at 2.7 Å resolution of a recombinant single-chain Fv fragment of dues at the antigen binding site was observed in the unliganded Fab crystal structure, which suggested that mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions electrostatic forces play an important role in the interaction between mAb1696 and HIV Pr. observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-To investigate further the mechanism of HIV Pr inhibition by mAb1696, we have now expressed a single-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. chain Fv fragment (scFv) in E. coli, which contains both 1696 variable domains joined by a flexible linking peptide. Such scFv constructs, which retain the full antigen Conclusion: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the binding capacities, are the object of very active research. Firstly, they are of interest for structural studies design of inhibitors aimed at binding inactive monomeric species. because they usually yield crystals diffracting to higher resolution than the corresponding Fab fragments on

Proceedings of the National Academy of Sciences, 1993

Although antibodies are highly specific, cross-reactions are frequently observed. To understand t... more Although antibodies are highly specific, cross-reactions are frequently observed. To understand the molecular basis of this phenomenon, we studied the anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with several avian lysozymes, in some cases with a higher affinity (heteroclitic binding) than for HEL. We have determined the crystal structure of the Fv fragment of D11.15 complexed with pheasant egg lysozyme (PHL). In addition, we have determined the structure of PHL, Guinea fowl egg lysozyme, and Japanese quail egg lysozyme. Differences in the affinity of D11.15 for the lysozymes appear to result from sequence substitutions in these antigens at the interface with the antibody. More generally, cross-reactivity is seen to require a stereochemically permissive environment for the variant antigen residues at the antibody-antigen interface.

Nucleic Acids Research, 2006

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a righ... more The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged b-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.

Journal of Virology, 2005

Dengue fever is an important emerging public health concern, with several million viral infection... more Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5′-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion...

Journal of General Virology, 2009

The flavivirus envelope glycoprotein (E) is responsible for viral attachment and entry by membran... more The flavivirus envelope glycoprotein (E) is responsible for viral attachment and entry by membrane fusion. Its ectodomain is the primary target of the humoral immune response. In particular, the C-terminal Ig-like domain III of E, which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (mAb). 9F12, a mouse mAb raised against a dengue virus (DENV) serotype 2 recombinant domain III, cross-reacts with corresponding domains from the other three DENV serotypes and also with West Nile virus. mAb 9F12 binds with nanomolar affinity to a conserved epitope that maps to the viral surface comprising residues 305, 307, 310 and 330 of the E protein. mAb 9F12 neutralizes all four DENV serotypes in plaque reduction assays. We expressed a single-chain Fv from 9F12 that retains the binding activity of the parent mAb. Adsorption and fusion inhibition assays indicate that mAb 9F12 prevents early steps of viral entry. Its virus inhi...

Journal of General Virology, 2005

The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates en... more The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of β-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with ...

Journal of Biological Chemistry, 2009

Journal of Biological Chemistry, 2012

Background: DynE8 is an iterative polyketide synthase (PKS) that assembles polyketide intermediat... more Background: DynE8 is an iterative polyketide synthase (PKS) that assembles polyketide intermediates from acetate units derived from malonyl-CoA. Results: We report the first acyltransferase (AT DYN10) crystal structure for an iterative PKS. Conclusion: AT DYN10 protects the malonyl-enzyme, but not the acetyl-enzyme intermediate, from hydrolysis and facilitates the transfer of malonyl to the acyl carrier protein. Significance: This differs from the dual specificity exhibited by acyltransferases of mammalian FAS and other iterative PKSs. Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named AT DYN10) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 Å resolution unveils a ␣/␤ hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a ␣/␤ fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude ␣-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser 651 residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates. * This work is supported by the Ministry of Education of Singapore through Academic Research Council grants (to Z.-X. L.), Biomedical Research Council grant 08/1/22/19/589, and an Action Thematique et Incitative sur Programme from the CNRS (to J. L.). □ S This article contains supplemental Tables S1 and Figs. S1-S6. The atomic coordinates and structure factors (codes 4AMP and 4AMM) have been deposited in the Protein Data Bank,

Journal of Biological Chemistry, 2008

The Flavivirus NS5 protein possesses both (guanine-N7)methyltransferase and nucleoside-2-O methyl... more The Flavivirus NS5 protein possesses both (guanine-N7)methyltransferase and nucleoside-2-O methyltransferase activities required for sequential methylation of the cap structure present at the 5 end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-L-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)methyltransferase and nucleoside-2-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-L-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-L-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-L-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-L-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation. Cellular and many viral mRNAs contain a modified 5Ј-terminal guanosine "cap" structure covalently linked to the 5Ј end of

Journal of Bacteriology, 2009

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bac... more EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α) 8 barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa , we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant ( K m ) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease...