philippe Cuniasse | CEA - Academia.edu (original) (raw)

Papers by philippe Cuniasse

Research paper thumbnail of Structure d'oligodesoxynucleotides contenant des sites abasiques par résonance magnétique nucléaire

Les sites abasiques jouent un role dans le processus de mutagenese. Les resultats presentes dans ... more Les sites abasiques jouent un role dans le processus de mutagenese. Les resultats presentes dans cette etude concernent les consequences structurales de la presence de sites abasiques dans l'adn. Des oligonucleotides contenant des sites abasiques ont ete etudies par resonance magnetique nucleaire. Les resultats indiquent que la presence d'un site abasique n'est pas incompatibles avec le maintient de la geometrie b. En effet, les resultats obtenus pour l'oligonucleotide contenant une adenine face au site abasique montrent une forme b, l'adenine non appariee et le sucre du site abasique etant a l'interieur de l'helice. Toutefois la structure engendree par la presence d'un site abasique depend de la base qui fait face au site. Ainsi, une cytosine face a un site abasique est rejetee a l'interieur de l'helice d'adn. Le sucre du site abasique est egalement situe a l'exterieur de l'helice. Les resultats obtenus avec deux autres oligonucle...

Research paper thumbnail of Mechanism of MRX inhibition by Rif2 at telomeres

Nature Communications, 2021

Specific proteins present at telomeres ensure chromosome end stability, in large part through unk... more Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5’ end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its AT...

Research paper thumbnail of Grafting of functional motifs onto protein scaffolds identified by PDB screening - an efficient route to design optimizable protein binders

FEBS Journal, 2012

Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMP... more Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif-grafting approach. The motif corresponded to the four N-terminal residues of TIMP-2, a broad-spectrum protein inhibitor of MMPs. Scaffolds that are able to reproduce the functional topology of this motif were obtained by exhaustive screening of the Protein Data Bank (PDB) using STAMPS software (search for three-dimensional atom motifs in protein structures). Ten artificial protein binders were produced. The designed proteins bind catalytic sites of MMPs with affinities ranging from 450 nM to 450 lM prior to optimization. The crystal structure of one artificial binder in complex with the catalytic domain of MMP-12 showed that the inter-molecular interactions established by the functional motif in the artificial binder corresponded to those found in the MMP-14-TIMP-2 complex, albeit with some differences in geometry. Molecular dynamics simulations of the ten binders in complex with MMP-14 suggested that these scaffolds may allow partial reproduction of native inter-molecular interactions, but differences in geometry and stability may contribute to the lower affinity of the artificial protein binders compared to the natural protein binder. Nevertheless, these results show that the in silico design method used provides sets of protein binders that target a specific binding site with a good rate of success. This approach may constitute the first step of an efficient hybrid computational/experimental approach to protein binder design.

Research paper thumbnail of Development of Selective Inhibitors and Substrate of Matrix Metalloproteinase-12

Journal of Biological Chemistry, 2006

Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO 2-CH 2)-X... more Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO 2-CH 2)-Xaa-Yaa-Zaa-NH 2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with K i values of 0.19 and 4.4 nM toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa-Zaa positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr 239 and Lys 177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm.

Research paper thumbnail of An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

Nucleic Acids Research, 1987

We have determined the three-dimensional structure of a nonselfcoropleroentary nonanucleot ide du... more We have determined the three-dimensional structure of a nonselfcoropleroentary nonanucleot ide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assiggned by NOESY, COSY and 2DQF spectra in DjO and HjO. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to SO ras), and-front these Mere able to establish interproton distances •for the central part of the duplex. We propose a di-f-ferent strategy-for proton-proton distance deterrainations which takes into account the observed variations in correlation times-for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotIde structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DMA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tra of this base being the same as that of the A>T base pairs in the sane duplex.

Research paper thumbnail of Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 2 antigenic polymorphism

We report in this study the molecular engineering of nanobodies that bind with picomolar affinity... more We report in this study the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domains (RBD) and are highly neutralizing. We applied Deep Mutational Engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of rec...

Research paper thumbnail of Inhibiteurs selectifs de site n-terminal de l'eca

L'invention concerne des derives de peptides utilisables comme inhibiteur selectif du site N-... more L'invention concerne des derives de peptides utilisables comme inhibiteur selectif du site N-terminal de l'enzyme de conversion de l'angiotensine humaine. Les derives comportent la sequence d'acides amines de formule suivante: -Asp-Phe-(PO2CH2)-Ala-Xaa'- dans laquelle: (PO2CH2) indique que la liaison peptidique (CONH) entre Phe et Ala a ete remplacee par la liaison phosphonique PO2CH2; et Xaa' represente un residu d'acide amine. Ils peuvent etre utilises dans des compositions pharmaceutiques, notamment pour proteger les cellules souches hematopoietiques de patients soumis a un traitement chimiotherapeutique ou radiotherapeutique agressif.

Research paper thumbnail of Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

Nucleic Acids Research

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitous... more Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosph...

Research paper thumbnail of The CTD of HpDprA, a DNA binding Winged Helix domain which do not bind dsDNA

with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:

Research paper thumbnail of The C-terminal domain of HpDprA is a DNA-binding winged helix domain that does not bind double-stranded DNA

The FEBS Journal

The winged helix domain of H. pylori DprA J. Lisboa et al.

Research paper thumbnail of Structures of biomolecular complexes by combination of NMR and cryoEM methods

Current Opinion in Structural Biology

CryoEM is presently providing structures of biocomplexes considered intractable to analysis by ot... more CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid-or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics. Modeling based on solid-state NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR became an established hybrid approach in structural biology that significantly contributes to our understanding of functional mechanisms in supramolecular assemblies. Highlights  CryoEM combined with NMR provided atomic models of biocomplexes.  NMR structures together with X-ray structures enabled interpretation of cryoEM maps.  Solid-state NMR and cryoEM resolved structures of helical filaments and fibrils.  These approaches validated and improved models built de novo using cryoEM maps.  NMR structures were compared with cryoEM models to uncover assembly mechanisms.

Research paper thumbnail of High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

Scientific Reports, 2017

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the vir... more Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of ...

Research paper thumbnail of Erratum: High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

Scientific Reports, 2017

In the results section under subheading 'The C-terminal domain adopts an immunoglobulin fold in s... more In the results section under subheading 'The C-terminal domain adopts an immunoglobulin fold in solution', "A search for structurally similar proteins possessing using the Dali server identified many proteins containing an Ig-like fold, including immunoglobulin receptors, fibroblast growth factor receptors and the T-cell surface glycoprotein CD4. " should read: "A search for structurally similar proteins using the Dali server identified many proteins containing an Ig-like fold, including immunoglobulin receptors, fibroblast growth factor receptors and the T-cell surface glycoprotein CD4. " In Table 2 in the MgCl 2 column, the second T5∆dec value '1 mM' should read '0.1 mM' .

Research paper thumbnail of Cysteine-Free Efficient Technetium (Tc) or Rhenium (Re) Chelating Peptide Tags and Their Use

Research paper thumbnail of Phosphinic pseudo-peptides that may be used as matrix zinc metalloprotease inhibitors

Research paper thumbnail of Residues determining the specific binding of uncompetitive inhibitors to mammalian alkaline phosphatases

The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase... more The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling. In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor. The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.

Research paper thumbnail of Phosphinic pseudopeptides inhibitors of matrix metalloproteases

Research paper thumbnail of Inhibition Mechanism of Membrane Metalloprotease by an Exosite-Swiveling Conformational Antibody

Structure, 2015

Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MM... more Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells. D = 2.32 3 10 À9 M).

Research paper thumbnail of Cyclic peptides with a phosphinic bond: novel templates for the design of inhibitors of zinc metalloproteases?

Journal of Molecular Graphics, 1993

Research paper thumbnail of Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain

The FASEB Journal, 2013

Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tis... more Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14-26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended ␤-strand with Glu 40 or Tyr 46 inserted into the S 1 = specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase.-Stura, E. A., Visse, R., Cuniasse, P., Dive, V., Nagase, H. Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain.

Research paper thumbnail of Structure d'oligodesoxynucleotides contenant des sites abasiques par résonance magnétique nucléaire

Les sites abasiques jouent un role dans le processus de mutagenese. Les resultats presentes dans ... more Les sites abasiques jouent un role dans le processus de mutagenese. Les resultats presentes dans cette etude concernent les consequences structurales de la presence de sites abasiques dans l'adn. Des oligonucleotides contenant des sites abasiques ont ete etudies par resonance magnetique nucleaire. Les resultats indiquent que la presence d'un site abasique n'est pas incompatibles avec le maintient de la geometrie b. En effet, les resultats obtenus pour l'oligonucleotide contenant une adenine face au site abasique montrent une forme b, l'adenine non appariee et le sucre du site abasique etant a l'interieur de l'helice. Toutefois la structure engendree par la presence d'un site abasique depend de la base qui fait face au site. Ainsi, une cytosine face a un site abasique est rejetee a l'interieur de l'helice d'adn. Le sucre du site abasique est egalement situe a l'exterieur de l'helice. Les resultats obtenus avec deux autres oligonucle...

Research paper thumbnail of Mechanism of MRX inhibition by Rif2 at telomeres

Nature Communications, 2021

Specific proteins present at telomeres ensure chromosome end stability, in large part through unk... more Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5’ end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its AT...

Research paper thumbnail of Grafting of functional motifs onto protein scaffolds identified by PDB screening - an efficient route to design optimizable protein binders

FEBS Journal, 2012

Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMP... more Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif-grafting approach. The motif corresponded to the four N-terminal residues of TIMP-2, a broad-spectrum protein inhibitor of MMPs. Scaffolds that are able to reproduce the functional topology of this motif were obtained by exhaustive screening of the Protein Data Bank (PDB) using STAMPS software (search for three-dimensional atom motifs in protein structures). Ten artificial protein binders were produced. The designed proteins bind catalytic sites of MMPs with affinities ranging from 450 nM to 450 lM prior to optimization. The crystal structure of one artificial binder in complex with the catalytic domain of MMP-12 showed that the inter-molecular interactions established by the functional motif in the artificial binder corresponded to those found in the MMP-14-TIMP-2 complex, albeit with some differences in geometry. Molecular dynamics simulations of the ten binders in complex with MMP-14 suggested that these scaffolds may allow partial reproduction of native inter-molecular interactions, but differences in geometry and stability may contribute to the lower affinity of the artificial protein binders compared to the natural protein binder. Nevertheless, these results show that the in silico design method used provides sets of protein binders that target a specific binding site with a good rate of success. This approach may constitute the first step of an efficient hybrid computational/experimental approach to protein binder design.

Research paper thumbnail of Development of Selective Inhibitors and Substrate of Matrix Metalloproteinase-12

Journal of Biological Chemistry, 2006

Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO 2-CH 2)-X... more Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO 2-CH 2)-Xaa-Yaa-Zaa-NH 2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with K i values of 0.19 and 4.4 nM toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa-Zaa positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr 239 and Lys 177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm.

Research paper thumbnail of An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations

Nucleic Acids Research, 1987

We have determined the three-dimensional structure of a nonselfcoropleroentary nonanucleot ide du... more We have determined the three-dimensional structure of a nonselfcoropleroentary nonanucleot ide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assiggned by NOESY, COSY and 2DQF spectra in DjO and HjO. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to SO ras), and-front these Mere able to establish interproton distances •for the central part of the duplex. We propose a di-f-ferent strategy-for proton-proton distance deterrainations which takes into account the observed variations in correlation times-for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotIde structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DMA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tra of this base being the same as that of the A>T base pairs in the sane duplex.

Research paper thumbnail of Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 2 antigenic polymorphism

We report in this study the molecular engineering of nanobodies that bind with picomolar affinity... more We report in this study the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domains (RBD) and are highly neutralizing. We applied Deep Mutational Engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of rec...

Research paper thumbnail of Inhibiteurs selectifs de site n-terminal de l'eca

L'invention concerne des derives de peptides utilisables comme inhibiteur selectif du site N-... more L'invention concerne des derives de peptides utilisables comme inhibiteur selectif du site N-terminal de l'enzyme de conversion de l'angiotensine humaine. Les derives comportent la sequence d'acides amines de formule suivante: -Asp-Phe-(PO2CH2)-Ala-Xaa'- dans laquelle: (PO2CH2) indique que la liaison peptidique (CONH) entre Phe et Ala a ete remplacee par la liaison phosphonique PO2CH2; et Xaa' represente un residu d'acide amine. Ils peuvent etre utilises dans des compositions pharmaceutiques, notamment pour proteger les cellules souches hematopoietiques de patients soumis a un traitement chimiotherapeutique ou radiotherapeutique agressif.

Research paper thumbnail of Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

Nucleic Acids Research

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitous... more Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosph...

Research paper thumbnail of The CTD of HpDprA, a DNA binding Winged Helix domain which do not bind dsDNA

with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:

Research paper thumbnail of The C-terminal domain of HpDprA is a DNA-binding winged helix domain that does not bind double-stranded DNA

The FEBS Journal

The winged helix domain of H. pylori DprA J. Lisboa et al.

Research paper thumbnail of Structures of biomolecular complexes by combination of NMR and cryoEM methods

Current Opinion in Structural Biology

CryoEM is presently providing structures of biocomplexes considered intractable to analysis by ot... more CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid-or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics. Modeling based on solid-state NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR became an established hybrid approach in structural biology that significantly contributes to our understanding of functional mechanisms in supramolecular assemblies. Highlights  CryoEM combined with NMR provided atomic models of biocomplexes.  NMR structures together with X-ray structures enabled interpretation of cryoEM maps.  Solid-state NMR and cryoEM resolved structures of helical filaments and fibrils.  These approaches validated and improved models built de novo using cryoEM maps.  NMR structures were compared with cryoEM models to uncover assembly mechanisms.

Research paper thumbnail of High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

Scientific Reports, 2017

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the vir... more Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of ...

Research paper thumbnail of Erratum: High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

Scientific Reports, 2017

In the results section under subheading 'The C-terminal domain adopts an immunoglobulin fold in s... more In the results section under subheading 'The C-terminal domain adopts an immunoglobulin fold in solution', "A search for structurally similar proteins possessing using the Dali server identified many proteins containing an Ig-like fold, including immunoglobulin receptors, fibroblast growth factor receptors and the T-cell surface glycoprotein CD4. " should read: "A search for structurally similar proteins using the Dali server identified many proteins containing an Ig-like fold, including immunoglobulin receptors, fibroblast growth factor receptors and the T-cell surface glycoprotein CD4. " In Table 2 in the MgCl 2 column, the second T5∆dec value '1 mM' should read '0.1 mM' .

Research paper thumbnail of Cysteine-Free Efficient Technetium (Tc) or Rhenium (Re) Chelating Peptide Tags and Their Use

Research paper thumbnail of Phosphinic pseudo-peptides that may be used as matrix zinc metalloprotease inhibitors

Research paper thumbnail of Residues determining the specific binding of uncompetitive inhibitors to mammalian alkaline phosphatases

The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase... more The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling. In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor. The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.

Research paper thumbnail of Phosphinic pseudopeptides inhibitors of matrix metalloproteases

Research paper thumbnail of Inhibition Mechanism of Membrane Metalloprotease by an Exosite-Swiveling Conformational Antibody

Structure, 2015

Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MM... more Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells. D = 2.32 3 10 À9 M).

Research paper thumbnail of Cyclic peptides with a phosphinic bond: novel templates for the design of inhibitors of zinc metalloproteases?

Journal of Molecular Graphics, 1993

Research paper thumbnail of Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain

The FASEB Journal, 2013

Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tis... more Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14-26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended ␤-strand with Glu 40 or Tyr 46 inserted into the S 1 = specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase.-Stura, E. A., Visse, R., Cuniasse, P., Dive, V., Nagase, H. Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain.