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Martino Bolognesi - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoGlobin.
Biochimica et biophysica acta, 2013Co-Authors: Chiara Ciaccio, Alessandra Pesce, Sylvia Dewilde, Martino Bolognesi, Luc Moens, Paolo Ascenzi, L. Tilleman, Grazia R. Tundo, Laura Bertolacci, Marco NardiniAbstract:Abstract Functional and structural properties of protoGlobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the Globin Fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 A resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misFolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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The diversity of 2/2 (truncated) Globins.
Advances in microbial physiology, 2013Co-Authors: Alessandra Pesce, Martino Bolognesi, Marco NardiniAbstract:Abstract Small size Globins that have been defined as ‘truncated haemoGlobins’ or as ‘2/2 haemoGlobins’ have increasingly been discovered in microorganisms since the early 1990s. Analysis of amino acid sequences allowed to distinguish three groups that collect proteins with specific and common structural properties. All three groups display 3D structures that are based on four main α-helices, which are a subset of the conventional eight-helices Globin Fold. Specific features, such as the presence of protein matrix tunnels that are held to promote diffusion of functional ligands to/from the haem, distinguish members of the three groups. Haem distal sites vary for their accessibility, local structures, polarity, and ligand stabilization mechanisms, suggesting functional roles that are related to O 2 /NO chemistry. In a few cases, such activities have been proven in vitro and in vivo through deletion mutants. The issue of 2/2 haemoGlobin varied biological functions throughout the three groups remains however fully open.
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High Resolution Crystal Structures of the Cerebratulus Lacteus Mini-Hb in the Unligated and Carbomonoxy States.
International journal of molecular sciences, 2012Co-Authors: Francesca Germani, Sylvia Dewilde, Martino Bolognesi, Luc Moens, A. Pesce, Andrea Venturini, Marco NardiniAbstract:The nerve tissue mini-hemoGlobin from Cerebratulus lacteus (CerHb) displays an essential Globin Fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O2 dissociation and ultra-high O2 affinity. However, CerHb affinity for O2 is similar to that of mammalian myoGlobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O2 ligand, the overall binding schemes for CO and O2 are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the Folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.
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the hemoGlobins of the trematodes fasciola hepatica and paramphistomum epiclitum a molecular biological physico chemical kinetic and vaccination study
Protein Science, 2008Co-Authors: Sylvia Dewilde, Alessandra Pesce, Laurent Kiger, Michael C Marden, Sabine Van Doorslaer, M Nardini, Iulia A Ioanitescu, Kambiz Gilany, Jozef Vercruysse, Martino BolognesiAbstract:The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoGlobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain Globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the Globin Fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoGlobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.
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Exploring the molecular basis of heme coordination in human neuroGlobin.
Proteins, 2008Co-Authors: Alejandro D. Nadra, Alessandra Pesce, Martino Bolognesi, Marcelo A. Martí, Darío A. EstrinAbstract:NeuroGlobin (Ngb), a recently discovered ancient heme protein, presents the typical Globin Fold and is around 20% identical to myoGlobin (Mb). In contrast with Mb, however, its heme is hexacoordinated (6c). It is expressed in the nervous system and has been the subject of numerous investigations in the last years, but its function is still unclear. The proposed roles include oxygen transport, reactive oxygen species (ROS) detoxification, hypoxia protection, and redox state sensing. All proposed functions require distal histidine dissociation from the heme to yield a reactive iron. With the aim of understanding the 6c to 5c transition, we have performed molecular dynamics simulations for ferrous Ngb in the 6c, 5c, and oxy states. We also computed free energy profiles associated with the transition employing an advanced sampling technique. Finally, we studied the effect of the redox state of CysCD7 and CysD5, which are known to form a disulfide bridge. Our results show that protein oxidation promotes a stabilization of the pentacoordinated species, thus favoring the protein to adopt the more reactive state and supporting the existence of a molecular mechanism whereby O2 would be released under hypoxic conditions, thereby suggesting an O(2) storage function for Ngb. Taken together, our results provide structural information not available experimentally which may shed light on the protein proposed functions, particularly as a redox sensor.
Sylvia Dewilde - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoGlobin.
Biochimica et biophysica acta, 2013Co-Authors: Chiara Ciaccio, Alessandra Pesce, Sylvia Dewilde, Martino Bolognesi, Luc Moens, Paolo Ascenzi, L. Tilleman, Grazia R. Tundo, Laura Bertolacci, Marco NardiniAbstract:Abstract Functional and structural properties of protoGlobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the Globin Fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 A resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misFolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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High Resolution Crystal Structures of the Cerebratulus Lacteus Mini-Hb in the Unligated and Carbomonoxy States.
International journal of molecular sciences, 2012Co-Authors: Francesca Germani, Sylvia Dewilde, Martino Bolognesi, Luc Moens, A. Pesce, Andrea Venturini, Marco NardiniAbstract:The nerve tissue mini-hemoGlobin from Cerebratulus lacteus (CerHb) displays an essential Globin Fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O2 dissociation and ultra-high O2 affinity. However, CerHb affinity for O2 is similar to that of mammalian myoGlobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O2 ligand, the overall binding schemes for CO and O2 are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the Folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.
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Globins in Caenorhabditis elegans
IUBMB life, 2011Co-Authors: L. Tilleman, Luc Moens, David Hoogewijs, Jacques R Vanfleteren, Eva Geuens, Francesca Germani, Sasha De Henau, Bart P. Braeckman, Sylvia DewildeAbstract:Summary Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for Globins that are all transcribed. These Globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans Globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four Globins (GLB1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two Globins (GLB-5 and GLB-26), and overlays of the Globins were constructed to highlight the structural diversity among them. It is clear that although they all share the Globin Fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the Globin family of C. elegans and suggest potential functions. 2011 IUBMB IUBMB Life, 63(3): 166–174, 2011
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CytoGlobin conformations and disulfide bond formation: CytoGlobin conformations and disulfide bond formation
The FEBS journal, 2010Co-Authors: Christophe Lechauve, Sylvia Dewilde, Michael C Marden, Luc Moens, Cédric Chauvierre, Brian N. Green, Chantal Celier, Laurent KigerAbstract:The oligomeric state and kinetics of ligand binding were measured for wild-type cytoGlobin. CytoGlobin has the classical Globin Fold, with an extension at each extremity of about 20 residues. The extended length of cytoGlobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoGlobin behaves hydrodynamically like a tightly packed Globin with a greater flexibility of the N- and C-terminal regions. CytoGlobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoGlobin conformations, which may affect the observed oxygen affinity by an order of magnitude.
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the hemoGlobins of the trematodes fasciola hepatica and paramphistomum epiclitum a molecular biological physico chemical kinetic and vaccination study
Protein Science, 2008Co-Authors: Sylvia Dewilde, Alessandra Pesce, Laurent Kiger, Michael C Marden, Sabine Van Doorslaer, M Nardini, Iulia A Ioanitescu, Kambiz Gilany, Jozef Vercruysse, Martino BolognesiAbstract:The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoGlobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain Globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the Globin Fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoGlobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.
Alessandra Pesce - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoGlobin.
Biochimica et biophysica acta, 2013Co-Authors: Chiara Ciaccio, Alessandra Pesce, Sylvia Dewilde, Martino Bolognesi, Luc Moens, Paolo Ascenzi, L. Tilleman, Grazia R. Tundo, Laura Bertolacci, Marco NardiniAbstract:Abstract Functional and structural properties of protoGlobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the Globin Fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 A resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misFolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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The diversity of 2/2 (truncated) Globins.
Advances in microbial physiology, 2013Co-Authors: Alessandra Pesce, Martino Bolognesi, Marco NardiniAbstract:Abstract Small size Globins that have been defined as ‘truncated haemoGlobins’ or as ‘2/2 haemoGlobins’ have increasingly been discovered in microorganisms since the early 1990s. Analysis of amino acid sequences allowed to distinguish three groups that collect proteins with specific and common structural properties. All three groups display 3D structures that are based on four main α-helices, which are a subset of the conventional eight-helices Globin Fold. Specific features, such as the presence of protein matrix tunnels that are held to promote diffusion of functional ligands to/from the haem, distinguish members of the three groups. Haem distal sites vary for their accessibility, local structures, polarity, and ligand stabilization mechanisms, suggesting functional roles that are related to O 2 /NO chemistry. In a few cases, such activities have been proven in vitro and in vivo through deletion mutants. The issue of 2/2 haemoGlobin varied biological functions throughout the three groups remains however fully open.
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Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties.
BMC Biochemistry, 2010Co-Authors: Eva Geuens, Alessandra Pesce, Laurent Kiger, David Hoogewijs, L. Tilleman, Angela Fago, Sasha De Henau, Marco Nardini, Evi Vinck, Michael C MardenAbstract:BACKGROUND: The genome of the nematode Caenorhabditis elegans contains more than 30 putative Globin genes that all are transcribed. Although their translated amino acid sequences fit the Globin Fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative Globins. No information is available on the physicochemical properties and the in vivo expression. RESULTS: We expressed the Globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 --> Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three alpha-helical sandwich of vertebrate Globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other Globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This Globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. CONCLUSION: The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different Globin classes.
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the hemoGlobins of the trematodes fasciola hepatica and paramphistomum epiclitum a molecular biological physico chemical kinetic and vaccination study
Protein Science, 2008Co-Authors: Sylvia Dewilde, Alessandra Pesce, Laurent Kiger, Michael C Marden, Sabine Van Doorslaer, M Nardini, Iulia A Ioanitescu, Kambiz Gilany, Jozef Vercruysse, Martino BolognesiAbstract:The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoGlobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain Globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the Globin Fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoGlobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.
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Exploring the molecular basis of heme coordination in human neuroGlobin.
Proteins, 2008Co-Authors: Alejandro D. Nadra, Alessandra Pesce, Martino Bolognesi, Marcelo A. Martí, Darío A. EstrinAbstract:NeuroGlobin (Ngb), a recently discovered ancient heme protein, presents the typical Globin Fold and is around 20% identical to myoGlobin (Mb). In contrast with Mb, however, its heme is hexacoordinated (6c). It is expressed in the nervous system and has been the subject of numerous investigations in the last years, but its function is still unclear. The proposed roles include oxygen transport, reactive oxygen species (ROS) detoxification, hypoxia protection, and redox state sensing. All proposed functions require distal histidine dissociation from the heme to yield a reactive iron. With the aim of understanding the 6c to 5c transition, we have performed molecular dynamics simulations for ferrous Ngb in the 6c, 5c, and oxy states. We also computed free energy profiles associated with the transition employing an advanced sampling technique. Finally, we studied the effect of the redox state of CysCD7 and CysD5, which are known to form a disulfide bridge. Our results show that protein oxidation promotes a stabilization of the pentacoordinated species, thus favoring the protein to adopt the more reactive state and supporting the existence of a molecular mechanism whereby O2 would be released under hypoxic conditions, thereby suggesting an O(2) storage function for Ngb. Taken together, our results provide structural information not available experimentally which may shed light on the protein proposed functions, particularly as a redox sensor.
Luc Moens - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoGlobin.
Biochimica et biophysica acta, 2013Co-Authors: Chiara Ciaccio, Alessandra Pesce, Sylvia Dewilde, Martino Bolognesi, Luc Moens, Paolo Ascenzi, L. Tilleman, Grazia R. Tundo, Laura Bertolacci, Marco NardiniAbstract:Abstract Functional and structural properties of protoGlobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the Globin Fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 A resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misFolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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High Resolution Crystal Structures of the Cerebratulus Lacteus Mini-Hb in the Unligated and Carbomonoxy States.
International journal of molecular sciences, 2012Co-Authors: Francesca Germani, Sylvia Dewilde, Martino Bolognesi, Luc Moens, A. Pesce, Andrea Venturini, Marco NardiniAbstract:The nerve tissue mini-hemoGlobin from Cerebratulus lacteus (CerHb) displays an essential Globin Fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O2 dissociation and ultra-high O2 affinity. However, CerHb affinity for O2 is similar to that of mammalian myoGlobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O2 ligand, the overall binding schemes for CO and O2 are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the Folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.
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Globins in Caenorhabditis elegans
IUBMB life, 2011Co-Authors: L. Tilleman, Luc Moens, David Hoogewijs, Jacques R Vanfleteren, Eva Geuens, Francesca Germani, Sasha De Henau, Bart P. Braeckman, Sylvia DewildeAbstract:Summary Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for Globins that are all transcribed. These Globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans Globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four Globins (GLB1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two Globins (GLB-5 and GLB-26), and overlays of the Globins were constructed to highlight the structural diversity among them. It is clear that although they all share the Globin Fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the Globin family of C. elegans and suggest potential functions. 2011 IUBMB IUBMB Life, 63(3): 166–174, 2011
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CytoGlobin conformations and disulfide bond formation: CytoGlobin conformations and disulfide bond formation
The FEBS journal, 2010Co-Authors: Christophe Lechauve, Sylvia Dewilde, Michael C Marden, Luc Moens, Cédric Chauvierre, Brian N. Green, Chantal Celier, Laurent KigerAbstract:The oligomeric state and kinetics of ligand binding were measured for wild-type cytoGlobin. CytoGlobin has the classical Globin Fold, with an extension at each extremity of about 20 residues. The extended length of cytoGlobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoGlobin behaves hydrodynamically like a tightly packed Globin with a greater flexibility of the N- and C-terminal regions. CytoGlobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoGlobin conformations, which may affect the observed oxygen affinity by an order of magnitude.
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Archaeal ProtoGlobin Structure Indicates New Ligand Diffusion Paths and Modulation of Haem-Reactivity.
EMBO reports, 2008Co-Authors: Marco Nardini, Alessandra Pesce, Sylvia Dewilde, Paolo Ascenzi, Jennifer A. Saito, Maqsudul Alam, Liesbet Thijs, M. Coletta, Chiara Ciaccio, Luc MoensAbstract:The structural adaptability of the Globin Fold has been highlighted by the recent discovery of the 2-on-2 haemoGlobins, of neuroGlobin and cytoGlobin. ProtoGlobin from Methanosarcina acetivorans C2A—a strictly anaerobic methanogenic Archaea—is, to the best of our knowledge, the latest entry adding new variability and functional complexity to the haemoGlobin (Hb) superfamily. Here, we report the 1.3 A crystal structure of oxygenated M. acetivorans protoGlobin, together with the first insight into its ligand-binding properties. We show that, contrary to all known Globins, protoGlobin-specific loops and an amino-terminal extension completely bury the haem within the protein matrix. Access of O2, CO and NO to the haem is granted by the protoGlobin-specific apolar tunnels reaching the haem distal site from locations at the B/G and B/E helix interfaces. Functionally, M. acetivorans dimeric protoGlobin shows a selectivity ratio for O2/CO binding to the haem that favours O2 ligation and anticooperativity in ligand binding. Both properties are exceptional within the Hb superfamily.
Paolo Ascenzi - One of the best experts on this subject based on the ideXlab platform.
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Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoGlobin.
Biochimica et biophysica acta, 2013Co-Authors: Chiara Ciaccio, Alessandra Pesce, Sylvia Dewilde, Martino Bolognesi, Luc Moens, Paolo Ascenzi, L. Tilleman, Grazia R. Tundo, Laura Bertolacci, Marco NardiniAbstract:Abstract Functional and structural properties of protoGlobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the Globin Fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 A resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misFolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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Archaeal ProtoGlobin Structure Indicates New Ligand Diffusion Paths and Modulation of Haem-Reactivity.
EMBO reports, 2008Co-Authors: Marco Nardini, Alessandra Pesce, Sylvia Dewilde, Paolo Ascenzi, Jennifer A. Saito, Maqsudul Alam, Liesbet Thijs, M. Coletta, Chiara Ciaccio, Luc MoensAbstract:The structural adaptability of the Globin Fold has been highlighted by the recent discovery of the 2-on-2 haemoGlobins, of neuroGlobin and cytoGlobin. ProtoGlobin from Methanosarcina acetivorans C2A—a strictly anaerobic methanogenic Archaea—is, to the best of our knowledge, the latest entry adding new variability and functional complexity to the haemoGlobin (Hb) superfamily. Here, we report the 1.3 A crystal structure of oxygenated M. acetivorans protoGlobin, together with the first insight into its ligand-binding properties. We show that, contrary to all known Globins, protoGlobin-specific loops and an amino-terminal extension completely bury the haem within the protein matrix. Access of O2, CO and NO to the haem is granted by the protoGlobin-specific apolar tunnels reaching the haem distal site from locations at the B/G and B/E helix interfaces. Functionally, M. acetivorans dimeric protoGlobin shows a selectivity ratio for O2/CO binding to the haem that favours O2 ligation and anticooperativity in ligand binding. Both properties are exceptional within the Hb superfamily.
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Structural determinants in the group III truncated hemoGlobin from Campylobacter jejuni.
The Journal of biological chemistry, 2006Co-Authors: Marco Nardini, Alessandra Pesce, Paolo Ascenzi, Michel Guertin, Marie Labarre, Christian Richard, Alessandro Bolli, Martino BolognesiAbstract:Abstract Truncated hemoGlobins (trHbs) constitute a distinct lineage in the Globin superfamily, distantly related in size and Fold to myoGlobin and monomeric hemoGlobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified Globin Fold, essentially composed of a 2-on-2 α-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-A resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb Fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific Fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.
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Structural bases for heme binding and diatomic ligand recognition in truncated hemoGlobins.
Journal of inorganic biochemistry, 2005Co-Authors: Mario Milani, Alessandra Pesce, Sylvia Dewilde, Paolo Ascenzi, Marco Nardini, Michel Guertin, Hugues Ouellet, Yannick Ouellet, Alessio Bocedi, Luc MoensAbstract:Abstract Truncated hemoGlobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoGlobin (Hb) superfamily. TrHbs display amino acid sequences 20–40 residues shorter than classical (non)vertebrate Hbs and myoGlobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 α-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 α-helical Globin Fold, achieved through deletion/truncation of α-helices and specific residue substitutions. Despite their ‘minimal’ polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O 2 , CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.
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Mapping protein matrix cavities in human cytoGlobin through Xe atom binding
Biochemical and biophysical research communications, 2004Co-Authors: Daniele De Sanctis, Thorsten Burmester, Alessandra Pesce, Sylvia Dewilde, Thomas Hankeln, Luc Moens, Paolo Ascenzi, Martino BolognesiAbstract:Abstract CytoGlobin is the fourth recognized Globin type, almost ubiquitously distributed in human tissues; its function is still poorly understood. CytoGlobin displays a core region of about 150 residues, structurally related to hemoGlobin and myoGlobin, and two extra segments, about 20 residues each, at the N- and C-termini. The core region hosts a large apolar cavity, held to provide a ligand diffusion pathway to/from the heme, and/or ligand temporary docking sites. Here we report the crystal structure (2.4 A resolution, R -factor 19.1%) of a human cytoGlobin mutant bearing the CysB2(38) → Ser and CysE9(83) → Ser substitutions (CYGB*), treated under pressurized xenon. Three Xe atoms bind to the heme distal site region of CYGB* mapping the protein matrix apolar cavity. Despite the conserved Globin Fold, the cavity found in CYGB* is structured differently from those recognized to play a functional role in myoGlobin, neuroGlobin, truncated hemoGlobins, and Cerebratulus lacteus mini-hemoGlobin.