Mario Bianchet - Academia.edu (original) (raw)

Papers by Mario Bianchet

Nature Communications, 2014

Voltage-gated sodium channels (Na(v)) underlie the rapid upstroke of action potentials in excitab... more Voltage-gated sodium channels (Na(v)) underlie the rapid upstroke of action potentials in excitable tissues. Binding of channel-interactive proteins is essential for controlling fast and long-term inactivation. In the structure of the complex of the carboxy-terminal portion of Na(v)1.5 (CTNa(v)1.5) with calmodulin (CaM)-Mg(2+) reported here, both CaM lobes interact with the CTNa(v)1.5. On the basis of the differences between this structure and that of an inactivated complex, we propose that the structure reported here represents a non-inactivated state of the CTNa(v), that is, the state that is poised for activation. Electrophysiological characterization of mutants further supports the importance of the interactions identified in the structure. Isothermal titration calorimetry experiments show that CaM binds to CTNa(v)1.5 with high affinity. The results of this study provide unique insights into the physiological activation and the pathophysiology of Na(v) channels.

Molecular Membrane Biology, 2003

To couple the energy present in the electrochemical proton gradient, established across the mitoc... more To couple the energy present in the electrochemical proton gradient, established across the mitochondrial membrane by the respiratory chain, to the formation of ATP from ADP and Pi, ATP-synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha, beta). These changes are induced by the rotation of the gamma subunit driven by the translocation of protons through the c subunit of the membrane portion of the enzyme. During this process, the F1-portion of the ATP-synthase adopts at least two major conformations depending on the occupancy of the beta subunits: one with two nucleotides, the other with three. In the two-nucleotide structure, the empty beta subunit adopts an open conformation that is highly different from the other conformations of beta subunits: tight, loose and closed. The three-dimensional structures of the F1-ATPase in each of these two major conformations provide a framework for understanding the mechanism of energy coupling by the enzyme. The energetics associated with two different models of the reaction steps, analysed using molecular dynamics calculations, show that three-nucleotide intermediates do not occur in configurations with an open beta subunit; instead, they are stabilized by completing a jaw-like motion that closes the beta subunit around the nucleotide. Consequently, the energy driven, major conformational change takes place with the beta subunits in the tight, loose and closed conformation.

Proceedings of The National Academy of Sciences, 1995

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diapho... more Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C

Nature Structural Biology, 1996

Odorant binding protein (OBP) is the major odorant binding component of mammalian nasal mucosa. T... more Odorant binding protein (OBP) is the major odorant binding component of mammalian nasal mucosa. The two structures of bovine OBP reported in this paper (one crystallized as purified and one soaked in the presence of a selenium-containing odorant) show that:(i) the OBP ...

Nature Structural Biology, 2001

ADP-ribose (adenosine 5′-diphosphoribose, ADPR) is produced enzymatically as part of the turnover... more ADP-ribose (adenosine 5′-diphosphoribose, ADPR) is produced enzymatically as part of the turnover of NAD + , cyclic ADPR and poly ADPR. High intracellular levels of ADPR can result in nonenzymatic ADP-ribosylation of proteins, a deleterious process that inactivates enzymes and interferes with recognition processes that rely on enzymatic ADP-ribosylation. For example, actin polymerization is inhibited by nonenzymatic ADP-ribosylation at a cysteine residue 1 . In addition, histone H1 modified by ADPribosylation could serve as a site of elongation for polyADP-ribose polymerase 2 . Enzymatic ADP-ribosylations can also have deleterious effects; for example, they induce apoptosis in some systems 3,4 and mediate the cytotoxic effect of bacterial toxins 5 . Clearly, the control of ADP-ribose levels is essential for cellular maintenance.

Glycobiology, 2002

Galectins, a family of soluble β-galactosyl-binding lectins, are believed to mediate cell-cell an... more Galectins, a family of soluble β-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydratebinding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galβ1,3GlcNAc, and type 2, Galβ1,4GlcNAc; Tα, Galβ1,3GalNAcα; Tβ, Galβ1,3GalNAcβ) and gangliosides containing the Tβ structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu 67 with both axial and equatorial -OH at C-3 of GlcNAc (in Galβ1,4GlcNAc) or at C-4 of GalNAc (in Galβ1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.

Vitamins & Hormones, 2008

Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many qu... more Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a ping-pong mechanism in which the NAD(P)H and the substrate occupy alternatively overlapping regions of the same binding site and participate in a double hydride transfer: one from NAD(P)H to the FAD of the enzyme, and one from the FADH(2) of the enzyme to the quinone substrate. The main function of QR1 is probably the detoxification of dietary quinones but it may also contribute to the reduction of vitamin K for its involvement in blood coagulation. In addition, the same reaction that QR1 uses in the detoxification of quinones, activates some compounds making them cytotoxic. Since QR1 is elevated in many tumors, this property has encouraged the development of chemotherapeutic compounds that become cytotoxic after reduction by QR1. The structures of QR1 alone, and in complexes with substrates, inhibitors, and chemotherapeutic prodrugs, combined with biochemical and mechanistic studies have provided invaluable insight into the mechanism of the enzyme as well as suggestions for the improvements of the chemotherapeutic prodrugs. Similar information is beginning to accumulate about another related enzyme, QR2.

Neurobiology of disease, 2013

Human immunodeficiency virus-1 (HIV)-associated neurocognitive disorder (HAND) is a neurodegenera... more Human immunodeficiency virus-1 (HIV)-associated neurocognitive disorder (HAND) is a neurodegenerative disease for which there is no available neuroprotective therapy. Viral proteins, such as Tat, have been implicated as agents of neurotoxicity via multiple mechanisms, including effects by directly binding to the NMDA receptor. We evaluated the ability of the immune response against Tat to modulate neurotoxicity at glutamate receptors. Neurotoxicity was measured in primary neuronal-glial cultures and in hippocampal slice cultures. We used immunoprecipitation experiments to demonstrate interaction between Tat, NMDA receptor, and anti-Tat antibody. Using known structures of Tat and NMDA receptors, we developed a model of their interactions. Antibodies to Tat attenuated Tat-mediated neurotoxicity. Interestingly, Tat immune complexes also blocked neurotoxicity caused by NMDA receptor agonists but not kainate/AMPA receptor agonists. Neither Tat nor antibody alone blocked the excitotoxic e...

Journal of Bioenergetics and Biomembranes, 1997

Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the t... more Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR

Structure, 2004

GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to... more GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction. Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism. Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage. Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A.

Structure, 2007

Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing th... more Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 Å resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.

Proteins: Structure, Function, and Genetics, 2000

Galectin-1, S-type ␤-galactosylbinding lectins present in vertebrate and invertebrate species, ar... more Galectin-1, S-type ␤-galactosylbinding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high-resolution crystal structures of B. arenarum galectin-1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions.

Proceedings of the National Academy of Sciences, 2000

Abbreviations: DQ, duroquinone; QR, quinone reductase; QR1, NAD(P)H:quinone acceptor oxidoreducta... more Abbreviations: DQ, duroquinone; QR, quinone reductase; QR1, NAD(P)H:quinone acceptor oxidoreductase type 1; rQR1, rat QR1; hQR1, human QR1; mQR1, mouse QR1; CB, Cibacron Blue; apo, apoenzyme.

Proceedings of the National Academy of Sciences, 2005

During development, neurons are guided to their targets by shortand long-range attractive and rep... more During development, neurons are guided to their targets by shortand long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL fd) determined by x-ray diffraction to 2.0 Å resolution. The structure shows that MICAL fd is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICAL fd is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H 2O2 (Km,NAPDH ‫؍‬ 222 M; kcat ‫؍‬ 77 sec ؊1 ), a molecule with known signaling properties. We propose that the H 2O2 produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.

Nucleic Acids Research, 2006

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for... more Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

Nature Chemical Biology, 2009

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to ... more The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high-affinity inhibitors. Ideally, this strategy would use linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized owing to limitations in linker chemistry. Here we systematically explore the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase. Analysis of the free energies of binding in combination with cocrystal structures shows that the flexibility and strain of a given linker can have a substantial impact on binding affinity even when the binding fragments are optimally positioned. Such effects are not apparent from inspection of structures and underscore the importance of linker optimization in fragment-based drug discovery efforts.

Nature, 2007

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extr... more The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

Molecular Membrane Biology, 2003

To couple the energy present in the electrochemical proton gradient, established across the mitoc... more To couple the energy present in the electrochemical proton gradient, established across the mitochondrial membrane by the respiratory chain, to the formation of ATP from ADP and Pi, ATP-synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha, beta). These changes are induced by the rotation of the gamma subunit driven by the translocation of protons through the c subunit of the membrane portion of the enzyme. During this process, the F1-portion of the ATP-synthase adopts at least two major conformations depending on the occupancy of the beta subunits: one with two nucleotides, the other with three. In the two-nucleotide structure, the empty beta subunit adopts an open conformation that is highly different from the other conformations of beta subunits: tight, loose and closed. The three-dimensional structures of the F1-ATPase in each of these two major conformations provide a framework for understanding the mechanism of energy coupling by the enzyme. The energetics associated with two different models of the reaction steps, analysed using molecular dynamics calculations, show that three-nucleotide intermediates do not occur in configurations with an open beta subunit; instead, they are stabilized by completing a jaw-like motion that closes the beta subunit around the nucleotide. Consequently, the energy driven, major conformational change takes place with the beta subunits in the tight, loose and closed conformation.

Nature Communications, 2014

Voltage-gated sodium channels (Na(v)) underlie the rapid upstroke of action potentials in excitab... more Voltage-gated sodium channels (Na(v)) underlie the rapid upstroke of action potentials in excitable tissues. Binding of channel-interactive proteins is essential for controlling fast and long-term inactivation. In the structure of the complex of the carboxy-terminal portion of Na(v)1.5 (CTNa(v)1.5) with calmodulin (CaM)-Mg(2+) reported here, both CaM lobes interact with the CTNa(v)1.5. On the basis of the differences between this structure and that of an inactivated complex, we propose that the structure reported here represents a non-inactivated state of the CTNa(v), that is, the state that is poised for activation. Electrophysiological characterization of mutants further supports the importance of the interactions identified in the structure. Isothermal titration calorimetry experiments show that CaM binds to CTNa(v)1.5 with high affinity. The results of this study provide unique insights into the physiological activation and the pathophysiology of Na(v) channels.

Molecular Membrane Biology, 2003

To couple the energy present in the electrochemical proton gradient, established across the mitoc... more To couple the energy present in the electrochemical proton gradient, established across the mitochondrial membrane by the respiratory chain, to the formation of ATP from ADP and Pi, ATP-synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha, beta). These changes are induced by the rotation of the gamma subunit driven by the translocation of protons through the c subunit of the membrane portion of the enzyme. During this process, the F1-portion of the ATP-synthase adopts at least two major conformations depending on the occupancy of the beta subunits: one with two nucleotides, the other with three. In the two-nucleotide structure, the empty beta subunit adopts an open conformation that is highly different from the other conformations of beta subunits: tight, loose and closed. The three-dimensional structures of the F1-ATPase in each of these two major conformations provide a framework for understanding the mechanism of energy coupling by the enzyme. The energetics associated with two different models of the reaction steps, analysed using molecular dynamics calculations, show that three-nucleotide intermediates do not occur in configurations with an open beta subunit; instead, they are stabilized by completing a jaw-like motion that closes the beta subunit around the nucleotide. Consequently, the energy driven, major conformational change takes place with the beta subunits in the tight, loose and closed conformation.

Proceedings of The National Academy of Sciences, 1995

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diapho... more Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C

Nature Structural Biology, 1996

Odorant binding protein (OBP) is the major odorant binding component of mammalian nasal mucosa. T... more Odorant binding protein (OBP) is the major odorant binding component of mammalian nasal mucosa. The two structures of bovine OBP reported in this paper (one crystallized as purified and one soaked in the presence of a selenium-containing odorant) show that:(i) the OBP ...

Nature Structural Biology, 2001

ADP-ribose (adenosine 5′-diphosphoribose, ADPR) is produced enzymatically as part of the turnover... more ADP-ribose (adenosine 5′-diphosphoribose, ADPR) is produced enzymatically as part of the turnover of NAD + , cyclic ADPR and poly ADPR. High intracellular levels of ADPR can result in nonenzymatic ADP-ribosylation of proteins, a deleterious process that inactivates enzymes and interferes with recognition processes that rely on enzymatic ADP-ribosylation. For example, actin polymerization is inhibited by nonenzymatic ADP-ribosylation at a cysteine residue 1 . In addition, histone H1 modified by ADPribosylation could serve as a site of elongation for polyADP-ribose polymerase 2 . Enzymatic ADP-ribosylations can also have deleterious effects; for example, they induce apoptosis in some systems 3,4 and mediate the cytotoxic effect of bacterial toxins 5 . Clearly, the control of ADP-ribose levels is essential for cellular maintenance.

Glycobiology, 2002

Galectins, a family of soluble β-galactosyl-binding lectins, are believed to mediate cell-cell an... more Galectins, a family of soluble β-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydratebinding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galβ1,3GlcNAc, and type 2, Galβ1,4GlcNAc; Tα, Galβ1,3GalNAcα; Tβ, Galβ1,3GalNAcβ) and gangliosides containing the Tβ structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu 67 with both axial and equatorial -OH at C-3 of GlcNAc (in Galβ1,4GlcNAc) or at C-4 of GalNAc (in Galβ1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.

Vitamins & Hormones, 2008

Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many qu... more Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a ping-pong mechanism in which the NAD(P)H and the substrate occupy alternatively overlapping regions of the same binding site and participate in a double hydride transfer: one from NAD(P)H to the FAD of the enzyme, and one from the FADH(2) of the enzyme to the quinone substrate. The main function of QR1 is probably the detoxification of dietary quinones but it may also contribute to the reduction of vitamin K for its involvement in blood coagulation. In addition, the same reaction that QR1 uses in the detoxification of quinones, activates some compounds making them cytotoxic. Since QR1 is elevated in many tumors, this property has encouraged the development of chemotherapeutic compounds that become cytotoxic after reduction by QR1. The structures of QR1 alone, and in complexes with substrates, inhibitors, and chemotherapeutic prodrugs, combined with biochemical and mechanistic studies have provided invaluable insight into the mechanism of the enzyme as well as suggestions for the improvements of the chemotherapeutic prodrugs. Similar information is beginning to accumulate about another related enzyme, QR2.

Neurobiology of disease, 2013

Human immunodeficiency virus-1 (HIV)-associated neurocognitive disorder (HAND) is a neurodegenera... more Human immunodeficiency virus-1 (HIV)-associated neurocognitive disorder (HAND) is a neurodegenerative disease for which there is no available neuroprotective therapy. Viral proteins, such as Tat, have been implicated as agents of neurotoxicity via multiple mechanisms, including effects by directly binding to the NMDA receptor. We evaluated the ability of the immune response against Tat to modulate neurotoxicity at glutamate receptors. Neurotoxicity was measured in primary neuronal-glial cultures and in hippocampal slice cultures. We used immunoprecipitation experiments to demonstrate interaction between Tat, NMDA receptor, and anti-Tat antibody. Using known structures of Tat and NMDA receptors, we developed a model of their interactions. Antibodies to Tat attenuated Tat-mediated neurotoxicity. Interestingly, Tat immune complexes also blocked neurotoxicity caused by NMDA receptor agonists but not kainate/AMPA receptor agonists. Neither Tat nor antibody alone blocked the excitotoxic e...

Journal of Bioenergetics and Biomembranes, 1997

Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the t... more Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR

Structure, 2004

GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to... more GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction. Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism. Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage. Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A.

Structure, 2007

Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing th... more Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 Å resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.

Proteins: Structure, Function, and Genetics, 2000

Galectin-1, S-type ␤-galactosylbinding lectins present in vertebrate and invertebrate species, ar... more Galectin-1, S-type ␤-galactosylbinding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high-resolution crystal structures of B. arenarum galectin-1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions.

Proceedings of the National Academy of Sciences, 2000

Abbreviations: DQ, duroquinone; QR, quinone reductase; QR1, NAD(P)H:quinone acceptor oxidoreducta... more Abbreviations: DQ, duroquinone; QR, quinone reductase; QR1, NAD(P)H:quinone acceptor oxidoreductase type 1; rQR1, rat QR1; hQR1, human QR1; mQR1, mouse QR1; CB, Cibacron Blue; apo, apoenzyme.

Proceedings of the National Academy of Sciences, 2005

During development, neurons are guided to their targets by shortand long-range attractive and rep... more During development, neurons are guided to their targets by shortand long-range attractive and repulsive cues. MICAL, a large multidomain protein, is required for the combined action of semaphorins and plexins in axon guidance. Here, we present the structure of the N-terminal region of MICAL (MICAL fd) determined by x-ray diffraction to 2.0 Å resolution. The structure shows that MICAL fd is an FAD-containing module structurally similar to aromatic hydroxylases and amine oxidases. In addition, we present biochemical data that show that MICAL fd is a flavoenzyme that in the presence of NADPH reduces molecular oxygen to H 2O2 (Km,NAPDH ‫؍‬ 222 M; kcat ‫؍‬ 77 sec ؊1 ), a molecule with known signaling properties. We propose that the H 2O2 produced by this reaction may be one of the signaling molecules involved in axon guidance by MICAL.

Nucleic Acids Research, 2006

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for... more Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

Nature Chemical Biology, 2009

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to ... more The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high-affinity inhibitors. Ideally, this strategy would use linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized owing to limitations in linker chemistry. Here we systematically explore the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase. Analysis of the free energies of binding in combination with cocrystal structures shows that the flexibility and strain of a given linker can have a substantial impact on binding affinity even when the binding fragments are optimally positioned. Such effects are not apparent from inspection of structures and underscore the importance of linker optimization in fragment-based drug discovery efforts.

Nature, 2007

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extr... more The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

Molecular Membrane Biology, 2003

To couple the energy present in the electrochemical proton gradient, established across the mitoc... more To couple the energy present in the electrochemical proton gradient, established across the mitochondrial membrane by the respiratory chain, to the formation of ATP from ADP and Pi, ATP-synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha, beta). These changes are induced by the rotation of the gamma subunit driven by the translocation of protons through the c subunit of the membrane portion of the enzyme. During this process, the F1-portion of the ATP-synthase adopts at least two major conformations depending on the occupancy of the beta subunits: one with two nucleotides, the other with three. In the two-nucleotide structure, the empty beta subunit adopts an open conformation that is highly different from the other conformations of beta subunits: tight, loose and closed. The three-dimensional structures of the F1-ATPase in each of these two major conformations provide a framework for understanding the mechanism of energy coupling by the enzyme. The energetics associated with two different models of the reaction steps, analysed using molecular dynamics calculations, show that three-nucleotide intermediates do not occur in configurations with an open beta subunit; instead, they are stabilized by completing a jaw-like motion that closes the beta subunit around the nucleotide. Consequently, the energy driven, major conformational change takes place with the beta subunits in the tight, loose and closed conformation.