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The human brain hexacoordinated neuroglobin three-dimensional structure
Micron 35 (2004) 63–65 www.elsevier.com/locate/micron
The human brain hexacoordinated neuroglobin three-dimensional structure Alessandra Pescea, Sylvia Dewildeb, Marco Nardinia, Luc Moensb, Paolo Ascenzic, Thomas Hankelnd, Thorsten Burmestere, Martino Bolognesia,f,* a
Department of Physics, INFM and Centre for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, Genova I-16146, Italy b Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp B-2610, Belgium c Department of Biology, University ‘Roma Tre’, Viale Guglielmo Marconi 446, Roma I-00146, Italy d Institute of Molecular Genetics, Johannes Gutenberg University of Mainz, Becherweg 32, Mainz D-55099, Germany e Institute of Zoology, Johannes Gutenberg University of Mainz, Mu¨llerweg 6, Mainz D-55099, Germany f Structural Biology Unit, National Institute for Cancer Research, Largo Rosanna Benzi 10, Genova I-16132, Italy
Abstract Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O2 supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a six-coordinated heme. O2 and CO bind to the heme-iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold, adapted to host the reversible bis-histidyl heme complex, and an elongated protein matrix cavity, held to facilitate O2 diffusion to the heme. The structure of neuroglobin suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily. q 2003 Elsevier Ltd. All rights reserved. Keywords: Globin fold; Heme hexacoordination; Neuroglobin; Oxygen affinity; Protein cavities
Neuroglobin (Ngb) has recently been identified in the vertebrate nervous system, as well as in some endocrine tissues and in the mammalian retina (Burmester et al., 2000; Trent et al., 2001; Schmidt et al., 2003), where it is thought to act as a hypoxia-inducible neuroprotective factor in hypoxic– ischemic injury such as stroke. Comparison of human neuroglobin (NGB) with vertebrate Mb or Hb shows less than 25% amino acid sequence identity, with the sequence conservation among Ngbs from different species being much higher than between orthologous Hbs and Mbs (Pesce et al., 2002a). Spectroscopic studies show that both ferrous deoxygenated and ferric Ngb forms display a hexacoordinated heme, where residues HisF8 and HisE7 are the fifth (proximal) and the sixth (distal) Fe ligands, respectively. To understand the structural bases of NGB function, here we present the three-dimensional structure of NGB in its * Corresponding author. Address: Department of Physics, INFM and Centre for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, Genova I-16146, Italy. Tel./fax: þ 39-10-5737-306. E-mail address: [email protected] (M. Bolognesi). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.013
˚ resolution, as the first hexacoordinated ferric form, at 1.95 A instance of a hexacoordinated hemeprotein observed in vertebrates (Pesce et al., 2003). Insight onto NGB structural and functional properties may offer hints for the development of new therapeutics, either enhancing its activity or regulating its cytosolic expression. The 3-D structure of NGB (bearing the Cys46 ! Gly, Cys55 ! Ser, Cys120 ! Ser mutations, for crystallization purposes: NGBp) was solved by means of MAD methods, based on the anomalous signal of the heme-iron atom. Diffraction data were collected at three wavelengths at the ID29 ESRF beam line (Grenoble, France), using one crystal of the monoclinic form previously described, which displays four NGBp chains per asymmetric unit (Pesce et al., 2002b). Refinement of the crystal structure converged at a general R-factor value of 17.8% (R-free 23.3%), for ˚ resolution range, with ideal data in the 40.0 – 1.95 A stereochemical parameters. The final model contains 4753 protein atoms, 160 ordered solvent atoms and seven sulphate anions. The protein fold in each NGBp chain conforms to the classical three-over-three a-helical sandwich of mammalian
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A. Pesce et al. / Micron 35 (2004) 63–65
Mb and Hb. For all the chains, the largest structural deviations are observed in their CD-D regions, and in the N-terminal half of the heme distal E-helices. In particular, the 44– 54 CD segments always protrudes from the protein ˚ relative to the structurally core, with deviations . 2.0 A equivalent region in MB. Two of the four independent NGBp molecules (A and D) display a partly disordered CD-D region; since the CD-D region hosts two out of the three Cys residues that have been mutated for crystallization purposes (Cys46 ! Gly and Cys55 ! Ser), it cannot be excluded that the mutations enhance the conformational disorder of the CD-D tract and its structural deviation from the corresponding region of MB. The conformational flexibility of the CD-D regions in NGBp may have functional roles in relation to the need for displacing the HisE7 ligand upon O2 binding. It may then be expected that in deoxygenated-pentacoordinated NGB, the distal E helix shifts away from the heme, leaving room for O2 diffusion to the heme distal site, and stabilization of the heme Fe-bound ligand through a hydrogen bond, as observed in many oxygenated Mbs or Hbs. Such a mechanism would imply that structural transition(s) required for removal of the endogenous His(64)E7 ligand reshape the distal site environment, promoting O2 diffusion to the heme, in keeping with the known high affinity of O2 for the NGB pentacoordinated species. Thus, the observed flexibility at the CD-D globin region may be a structural fingerprint of Hbs exploiting hexacoordination as a mechanism for the control of O2 diffusion to/from the heme. The NGBp distal site (Fig. 1A) is characterised by a coordination bond connecting the heme-Fe atom to the distal His(64)E7 NE2 atom. As a result of protein coordination on the heme distal site, the E-helix is pulled ˚ , relative to MB. The distal towards the heme by about 3 A site is therefore crowded by the apolar residues Phe(28)B10, ˚ from Phe(42)CD1, and Val(68)E11 (all falling at , 4.0 A His(64)E7), whereas Lys(67)E10 falls outside the heme crevice, being stabilised by electrostatic interaction with the heme propionates. The proximal His(96)F8 residue provides a coordination bond to the heme-Fe atom. Notably, the distal and the proximal His imidazoles display approximately orthogonal azimuthal orientations. The His(96)F8 imidazole ring is stabilized by a hydrogen bond between the histidyl ND1 atom and Leu(92)F4 O atom. ˚ 3) is present in A large protein matrix cavity (about 120 A the protein region located between the heme distal site and the EF interhelical hinge (Fig. 1B). The cavity is lined with hydrophobic residues provided by B, E, G and H helices. The cavity is closed to external solvent by the protein structure comprised between Val(71)E14 and Ala(74)E17, between Leu85 and Tyr88, and by the side chains of Leu(136)H11 and Tyr(137)H12. However, inspection of the intramolecular contacts indicates that, in a dynamic context,
Fig. 1. (A) View of the heme NGBp distal site region (grey ribbon and residues), showing the Fe-coordinated HisE7 residue, PheB10, PheCD1, TyrCD3, LysE10, ValE11 residues and the E helix, superimposed on the corresponding residues of MB (black ribbon and thin bonds). The NGBp coordination bond between HisE7 and the heme-Fe atom is highlighted by a dotted black line. The hydrogen bond and salt bridge stabilizing the heme A propionate is black dotted. (B) NGBp schematic fold, displaying the protein matrix cavity (black mesh), which stretches from the heme distal site towards the protein surface. The cavity surfaces displayed have been ˚ radius probe. defined by a 1.4 A
it may gain solvent access through side chain fluctuations in the residues listed above. Analysis of the structural data base shows that a similarly structured cavity is a unique feature of NGBp, not
A. Pesce et al. / Micron 35 (2004) 63–65
comparable with the four smaller cavities found in sperm whale Mb (mapped by Xe binding; Tilton et al., 1984), nor with the protein matrix tunnel observed in prokaryotic truncated Hbs (Milani et al., 2001; Wittenberg et al., 2002). ˚ 3 cavity The free-energy cost of maintaining a 120 A within the protein matrix relative to protein stability can be estimated in the order of 2– 4 kcal/mol. Conservation of ˚ 3 cavity in NGBp may therefore indicate its a . 100 A involvement in key functional roles, such as that of providing a facilitated O2 diffusion pathway to/from the heme, as an alternative to the distal site gating mechanism based on the HisE7 residue of Hbs and Mbs (Perutz, 1989). Alternatively, the NGBp protein matrix cavity may act as a docking/storage station for the ligand during the protein functional cycle, when the heme is in the hexacoordinated state.
References Burmester, T., Weich, B., Reinhardt, S., Hankeln, T., 2000. A vertebrate globin expressed in the brain. Nature 407, 520–523. Milani, M., Pesce, A., Ouellet, Y., Ascenzi, P., Guertin, M., Bolognesi, M., 2001. Mycobacterium tuberculosis hemoglobin-N displays a protein tunnel suited for O2 diffusion to the heme. EMBO J. 20, 3902–3909.
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Perutz, M.F., 1989. Myoglobin and hemoglobin: role of distal residues in reactions with haem ligands. Trends Biochem. Sci. 14, 42–44. Pesce, A., Bolognesi, M., Ascenzi, P., Bocedi, A., Dewilde, S., Moens, L., Hankeln, T., Burmester, T., 2002a. Neuroglobin and cytoglobin: fresh blood for the vertebrate globin family. EMBO Rep. 3, 1146–1151. Pesce, A., Nardini, M., Dewilde, S., Ascenzi, P., Burmester, T., Hankeln, T., Moens, L., Bolognesi, M., 2002b. Human neuroglobin: crystals and preliminary X-ray diffraction analysis. Acta Crystallogr. D58, 1848–1850. Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln, T., Burmester, T., Bolognesi, M., 2003. Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity. Structure 11, 1087–1095. Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U., Burmester, T., 2003. How does the eye breathe? Evidence for neuroglobinmediated oxygen supply of the vertebrate retina. J. Biol. Chem. 278, 1932–1935. Tilton, R.F. Jr., Kuntz, I.D. Jr., Petsko, G.A., 1984. Cavities in proteins: ˚ structure of a metmyoglobin – xenon complex solved at 1.9 A resolution. Biochemistry 23, 2849– 2857. Trent, J.T. III, Watts, R.A., Hargrove, M.S., 2001. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J. Biol. Chem. 276, 30106–30110. Wittenberg, J.B., Bolognesi, M., Wittenberg, B.A., Guertin, M., 2002. Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes and plants. J. Biol. Chem. 277, 871–874.
The human brain hexacoordinated neuroglobin three-dimensional structure Alessandra Pescea, Sylvia Dewildeb, Marco Nardinia, Luc Moensb, Paolo Ascenzic, Thomas Hankelnd, Thorsten Burmestere, Martino Bolognesia,f,* a
Department of Physics, INFM and Centre for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, Genova I-16146, Italy b Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp B-2610, Belgium c Department of Biology, University ‘Roma Tre’, Viale Guglielmo Marconi 446, Roma I-00146, Italy d Institute of Molecular Genetics, Johannes Gutenberg University of Mainz, Becherweg 32, Mainz D-55099, Germany e Institute of Zoology, Johannes Gutenberg University of Mainz, Mu¨llerweg 6, Mainz D-55099, Germany f Structural Biology Unit, National Institute for Cancer Research, Largo Rosanna Benzi 10, Genova I-16132, Italy
Abstract Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O2 supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a six-coordinated heme. O2 and CO bind to the heme-iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold, adapted to host the reversible bis-histidyl heme complex, and an elongated protein matrix cavity, held to facilitate O2 diffusion to the heme. The structure of neuroglobin suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily. q 2003 Elsevier Ltd. All rights reserved. Keywords: Globin fold; Heme hexacoordination; Neuroglobin; Oxygen affinity; Protein cavities
Neuroglobin (Ngb) has recently been identified in the vertebrate nervous system, as well as in some endocrine tissues and in the mammalian retina (Burmester et al., 2000; Trent et al., 2001; Schmidt et al., 2003), where it is thought to act as a hypoxia-inducible neuroprotective factor in hypoxic– ischemic injury such as stroke. Comparison of human neuroglobin (NGB) with vertebrate Mb or Hb shows less than 25% amino acid sequence identity, with the sequence conservation among Ngbs from different species being much higher than between orthologous Hbs and Mbs (Pesce et al., 2002a). Spectroscopic studies show that both ferrous deoxygenated and ferric Ngb forms display a hexacoordinated heme, where residues HisF8 and HisE7 are the fifth (proximal) and the sixth (distal) Fe ligands, respectively. To understand the structural bases of NGB function, here we present the three-dimensional structure of NGB in its * Corresponding author. Address: Department of Physics, INFM and Centre for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, Genova I-16146, Italy. Tel./fax: þ 39-10-5737-306. E-mail address: [email protected] (M. Bolognesi). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.013
˚ resolution, as the first hexacoordinated ferric form, at 1.95 A instance of a hexacoordinated hemeprotein observed in vertebrates (Pesce et al., 2003). Insight onto NGB structural and functional properties may offer hints for the development of new therapeutics, either enhancing its activity or regulating its cytosolic expression. The 3-D structure of NGB (bearing the Cys46 ! Gly, Cys55 ! Ser, Cys120 ! Ser mutations, for crystallization purposes: NGBp) was solved by means of MAD methods, based on the anomalous signal of the heme-iron atom. Diffraction data were collected at three wavelengths at the ID29 ESRF beam line (Grenoble, France), using one crystal of the monoclinic form previously described, which displays four NGBp chains per asymmetric unit (Pesce et al., 2002b). Refinement of the crystal structure converged at a general R-factor value of 17.8% (R-free 23.3%), for ˚ resolution range, with ideal data in the 40.0 – 1.95 A stereochemical parameters. The final model contains 4753 protein atoms, 160 ordered solvent atoms and seven sulphate anions. The protein fold in each NGBp chain conforms to the classical three-over-three a-helical sandwich of mammalian
64
A. Pesce et al. / Micron 35 (2004) 63–65
Mb and Hb. For all the chains, the largest structural deviations are observed in their CD-D regions, and in the N-terminal half of the heme distal E-helices. In particular, the 44– 54 CD segments always protrudes from the protein ˚ relative to the structurally core, with deviations . 2.0 A equivalent region in MB. Two of the four independent NGBp molecules (A and D) display a partly disordered CD-D region; since the CD-D region hosts two out of the three Cys residues that have been mutated for crystallization purposes (Cys46 ! Gly and Cys55 ! Ser), it cannot be excluded that the mutations enhance the conformational disorder of the CD-D tract and its structural deviation from the corresponding region of MB. The conformational flexibility of the CD-D regions in NGBp may have functional roles in relation to the need for displacing the HisE7 ligand upon O2 binding. It may then be expected that in deoxygenated-pentacoordinated NGB, the distal E helix shifts away from the heme, leaving room for O2 diffusion to the heme distal site, and stabilization of the heme Fe-bound ligand through a hydrogen bond, as observed in many oxygenated Mbs or Hbs. Such a mechanism would imply that structural transition(s) required for removal of the endogenous His(64)E7 ligand reshape the distal site environment, promoting O2 diffusion to the heme, in keeping with the known high affinity of O2 for the NGB pentacoordinated species. Thus, the observed flexibility at the CD-D globin region may be a structural fingerprint of Hbs exploiting hexacoordination as a mechanism for the control of O2 diffusion to/from the heme. The NGBp distal site (Fig. 1A) is characterised by a coordination bond connecting the heme-Fe atom to the distal His(64)E7 NE2 atom. As a result of protein coordination on the heme distal site, the E-helix is pulled ˚ , relative to MB. The distal towards the heme by about 3 A site is therefore crowded by the apolar residues Phe(28)B10, ˚ from Phe(42)CD1, and Val(68)E11 (all falling at , 4.0 A His(64)E7), whereas Lys(67)E10 falls outside the heme crevice, being stabilised by electrostatic interaction with the heme propionates. The proximal His(96)F8 residue provides a coordination bond to the heme-Fe atom. Notably, the distal and the proximal His imidazoles display approximately orthogonal azimuthal orientations. The His(96)F8 imidazole ring is stabilized by a hydrogen bond between the histidyl ND1 atom and Leu(92)F4 O atom. ˚ 3) is present in A large protein matrix cavity (about 120 A the protein region located between the heme distal site and the EF interhelical hinge (Fig. 1B). The cavity is lined with hydrophobic residues provided by B, E, G and H helices. The cavity is closed to external solvent by the protein structure comprised between Val(71)E14 and Ala(74)E17, between Leu85 and Tyr88, and by the side chains of Leu(136)H11 and Tyr(137)H12. However, inspection of the intramolecular contacts indicates that, in a dynamic context,
Fig. 1. (A) View of the heme NGBp distal site region (grey ribbon and residues), showing the Fe-coordinated HisE7 residue, PheB10, PheCD1, TyrCD3, LysE10, ValE11 residues and the E helix, superimposed on the corresponding residues of MB (black ribbon and thin bonds). The NGBp coordination bond between HisE7 and the heme-Fe atom is highlighted by a dotted black line. The hydrogen bond and salt bridge stabilizing the heme A propionate is black dotted. (B) NGBp schematic fold, displaying the protein matrix cavity (black mesh), which stretches from the heme distal site towards the protein surface. The cavity surfaces displayed have been ˚ radius probe. defined by a 1.4 A
it may gain solvent access through side chain fluctuations in the residues listed above. Analysis of the structural data base shows that a similarly structured cavity is a unique feature of NGBp, not
A. Pesce et al. / Micron 35 (2004) 63–65
comparable with the four smaller cavities found in sperm whale Mb (mapped by Xe binding; Tilton et al., 1984), nor with the protein matrix tunnel observed in prokaryotic truncated Hbs (Milani et al., 2001; Wittenberg et al., 2002). ˚ 3 cavity The free-energy cost of maintaining a 120 A within the protein matrix relative to protein stability can be estimated in the order of 2– 4 kcal/mol. Conservation of ˚ 3 cavity in NGBp may therefore indicate its a . 100 A involvement in key functional roles, such as that of providing a facilitated O2 diffusion pathway to/from the heme, as an alternative to the distal site gating mechanism based on the HisE7 residue of Hbs and Mbs (Perutz, 1989). Alternatively, the NGBp protein matrix cavity may act as a docking/storage station for the ligand during the protein functional cycle, when the heme is in the hexacoordinated state.
References Burmester, T., Weich, B., Reinhardt, S., Hankeln, T., 2000. A vertebrate globin expressed in the brain. Nature 407, 520–523. Milani, M., Pesce, A., Ouellet, Y., Ascenzi, P., Guertin, M., Bolognesi, M., 2001. Mycobacterium tuberculosis hemoglobin-N displays a protein tunnel suited for O2 diffusion to the heme. EMBO J. 20, 3902–3909.
65
Perutz, M.F., 1989. Myoglobin and hemoglobin: role of distal residues in reactions with haem ligands. Trends Biochem. Sci. 14, 42–44. Pesce, A., Bolognesi, M., Ascenzi, P., Bocedi, A., Dewilde, S., Moens, L., Hankeln, T., Burmester, T., 2002a. Neuroglobin and cytoglobin: fresh blood for the vertebrate globin family. EMBO Rep. 3, 1146–1151. Pesce, A., Nardini, M., Dewilde, S., Ascenzi, P., Burmester, T., Hankeln, T., Moens, L., Bolognesi, M., 2002b. Human neuroglobin: crystals and preliminary X-ray diffraction analysis. Acta Crystallogr. D58, 1848–1850. Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln, T., Burmester, T., Bolognesi, M., 2003. Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity. Structure 11, 1087–1095. Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U., Burmester, T., 2003. How does the eye breathe? Evidence for neuroglobinmediated oxygen supply of the vertebrate retina. J. Biol. Chem. 278, 1932–1935. Tilton, R.F. Jr., Kuntz, I.D. Jr., Petsko, G.A., 1984. Cavities in proteins: ˚ structure of a metmyoglobin – xenon complex solved at 1.9 A resolution. Biochemistry 23, 2849– 2857. Trent, J.T. III, Watts, R.A., Hargrove, M.S., 2001. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J. Biol. Chem. 276, 30106–30110. Wittenberg, J.B., Bolognesi, M., Wittenberg, B.A., Guertin, M., 2002. Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes and plants. J. Biol. Chem. 277, 871–874.