Structure, Vol. 11, 1087–1095, September, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0969-2126(03)00166-7 Human Brain Neuroglobin Structure Reveals a Distinct Mode of Controlling Oxygen Affinity Alessandra Pesce,1 Sylvia Dewilde,2 a wide and functionally diversified protein homology Marco Nardini,1 Luc Moens,2 superfamily. Paolo Ascenzi,3 Thomas Hankeln,4 Thorsten Burmester,5 and Martino Bolognesi1,6,* 1Department of Physics—INFM Introduction and Centre for Excellence in Biomedical Research Globins are small respiratory proteins that reversibly University of Genova bind O2 by means of an iron-containing porphyrin ring. Via Dodecaneso 33 Globin-like proteins have been identified in bacteria, I-16146 Genova plants, fungi, and animals (Hardison, 1996, 1998). Most Italy globins are held to sustain O2 supply to the aerobic 2 Department of Biomedical Sciences metabolism of the respiratory chain (Wittenberg, 1992; University of Antwerp Brunori, 1999; Weber and Vinogradov, 2001; Merx et al., Universiteitsplein 1 2002), although they have also been ascribed enzymatic B-2610 Antwerp functions (Minning et al., 1999; Brunori, 2001; Flo¨ gel et Belgium al., 2001). In man and other vertebrates, red blood cell 3 Department of Biology hemoglobin (Hb) transports O2 in the circulatory system. University ‘Roma Tre’ Myoglobin (Mb), typically found in the cardiac and stri- Viale Guglielmo Marconi 446 ated muscles, facilitates O2 diffusion and NO detoxifica- I-00146 Roma tion (Wittenberg, 1970; Perutz, 1979, 1990; Wittenberg Italy and Wittenberg, 1989; Hardison, 1996, 1998; Brunori, 4 Institute of Molecular Genetics 1999; 2001; Flo¨ gel et al., 2001). Neuroglobin (Ngb) has Johannes Gutenberg University of Mainz recently been identified in the vertebrate nervous sys- Becherweg 32 tem (Burmester et al., 2000; Trent et al., 2001). Further- D-55099 Mainz more, cytoglobin (Cygb) has been discovered through Germany genomic searches as a fourth globin type expressed 5 Institute of Zoology almost ubiquitously in human tissues (Burmester et al., Johannes Gutenberg University of Mainz 2002; Trent and Hargrove, 2002). Mu¨ llerweg 6 Initially identified in mouse and man (Burmester et al., D-55099 Mainz 2000), Ngb has subsequently been recognized also in Germany rat, pufferfish, and zebrafish (Awenius et al., 2001; Zhang 6 Structural Biology Unit et al., 2002), suggesting a widespread distribution National Institute for Cancer Research among the vertebrate species. Phylogenetic analyses Largo Rosanna Benzi 10 indicate a common ancient evolutionary origin of verte- I-16132 Genova brate Ngbs and invertebrate nerve globins (Burmester Italy et al., 2000; Pesce et al., 2002a). Comparison of human neuroglobin (NGB) with vertebrate Mb or Hb shows less than 25% amino acid sequence identity (Figure 1), the sequence conservation among Ngbs from different spe- Summary cies being much higher than between orthologous Hbs and Mbs (Burmester et al., 2000). The NGB amino acid Neuroglobin, mainly expressed in vertebrate brain and sequence (Figure 1) fits well into a globin alignment retina, is a recently identified member of the globin based on the conserved ␣ helices and the structural superfamily. Augmenting O2 supply, neuroglobin pro- motifs of the classical globin fold (Holm and Sander, motes survival of neurons upon hypoxic injury, poten- 1993; Bolognesi et al., 1997; Burmester et al., 2000; tially limiting brain damage. In the absence of exoge- Awenius et al., 2001; Pesce et al., 2002a). nous ligands, neuroglobin displays a hexacoordinated Ngb is expressed at the micromolar level in the verte- heme. O2 and CO bind to the heme iron, displacing the brate central and peripheral nervous systems, as well endogenous HisE7 heme distal ligand. Hexacoordi- as in some endocrine tissues (Burmester et al., 2000; nated human neuroglobin displays a classical globin Reuss et al., 2002). Particularly high Ngb concentration fold adapted to host the reversible bis-histidyl heme has been reported in the mammalian retina, one of the complex and an elongated protein matrix cavity, held highest-oxygen-consuming tissues of the body (Schmidt to facilitate O2 diffusion to the heme. The neuroglobin et al., 2003). The degree of NGB expression appears to structure suggests that the classical globin fold is en- vary in different areas of the human brain, where it is dowed with striking adaptability, indicating that hemo- thought to act as hypoxia-inducible neuroprotective fac- globin and myoglobin are just two examples within tor in hypoxic ischemic injury, such as stroke (Burmester et al., 2000; Moens and Dewilde, 2000). In fact, Ngb has been shown to promote the cell survival of a neuronal *Correspondence: [email protected] cell line kept under hypoxic conditions (Sun et al., 2001, Structure 1088 Figure 1. Structure-Based Sequence Alignment of NGB, MB, HB (␣ and ␤ Chains), CYGB, Rice Nonsymbiotic Hb, and C. lacteus Nerve Hb The top line depicts the span of ␣ helices in MB (blue), with the topological position assignments, as defined within the vertebrate globin fold. Sequence numbering (bottom line) refers to NGB. The heme Fe-coordinating residues HisE7 and HisF8 are marked in yellow, while apolar residues lining the protein matrix cavity in NGB and in CerHb are marked in blue. Residues making contacts with the heme are highlighted by green asterisks. 2003). Although these observations support an impor- Results and Discussion tant role of Ngb in the supply of O2 in nervous tissues, other functions, for example, as an O2-consuming en- The Overall Structure of NGB* zyme or an O2 sensor, are debated (Pesce et al., 2002a). The three-dimensional structure of NGB (bearing the Spectroscopic studies show that both ferrous deoxy- Cys46 → Gly, Cys55 → Ser, and Cys120 → Ser mutations genated and ferric Ngb forms display a hexacoordinated for crystallization purposes, NGB*) was solved by MAD heme, where residues HisF8 and HisE7 are the fifth methods based on the anomalous signal of the heme (proximal) and the sixth (distal) Fe ligands, respectively. iron atom. Diffraction data were collected at three wave- O2 reversibly displaces the endogenous HisE7 heme lengths at the ID29 ESRF beamline (Grenoble, France; ligand, leading to oxygenation of Ngb. The O2 affinity Table 1), with one crystal of the monoclinic form pre- for the pentacoordinated Ngb species is very high. How- viously described, which displays four NGB* chains per ever, because of HisE7/O2 competition for coordination asymmetric unit (Pesce et al., 2002b). Refinement of the to the heme Fe atom, the actual O2 affinity for Ngb is crystal structure converged at a general R factor value medium (i.e., P50 ca. 1 torr), closely similar to that of Mb of 17.8% (Rfree of 23.3%), for data in the 40.0–1.95 A˚ (Burmester et al., 2000; Dewilde et al., 2001; Pesce et resolution range, with ideal stereochemical parameters al., 2002a). (Engh and Huber, 1991; Laskowski et al., 1993). The final To understand the structural bases of NGB function, model contains 4753 protein atoms, 160 ordered solvent we present here the three-dimensional structure of NGB atoms, and 7 sulfate anions (Table 1). in its hexacoordinated ferric form, at 1.95 A˚ resolution, The protein fold in each NGB* chain (Figure 2A) con- as the first instance of a hexacoordinated heme protein forms to the classical three over three ␣-helical sand- observed in vertebrates. Our results reveal that a sub- wich of mammalian Mb and Hb (Holm and Sander, 1993; stantially conserved globin fold can support reversible Bolognesi et al., 1997), where the seven/eight ␣ helices heme hexacoordination through structural flexibility in building up the globin fold are conventionally labeled, A, the NGB region comprised between C and E helices. B, …, H, according to their sequential order (topological Additionally, a wide protein core cavity, enclosing part sites are numbered sequentially within each ␣ helix; of the heme distal site, may provide a ligand store/ Perutz, 1979). For a comparison, rmsd values of diffusion path whose functional role has no counterpart 1.18–1.43 A˚ are calculated (over 108 C␣ atom pairs) for in vertebrate Hbs or Mbs. the four independent NGB* chains, in structural overlays Human Neuroglobin Crystal Structure 1089 Table 1. Data Collection and Refinement Statistics for NGB* MAD Data Collection Statistics Absorption Peak Inflection Point Remote Wavelength (A˚ ) 1.738 1.740 0.980 Resolution (A˚ ) 30–2.5 30–2.5 30–2.2 Mosaicity (Њ) 1.2 1.2 1.2 Completeness (%) 98.6 (93.8)a 98.8 (93.5) 99.2 (96.4) Rmerge (%) 6.1 (14.5) 8.5 (18.4) 8.9 (15.6) Total reflections 65,453 65,352 196,480 Unique reflections 17,339 17,369 25,803 Redundancy 3.8 (3.1) 3.8 (3.1) 7.6 (6.2) Average I/␴(I) 9 (5) 6 (4) 5 (4) High-Resolution Data Collection Statistics Wavelength (A˚ ) 0.933 Resolution limit (A˚ ) 40–1.95 Mosaicity (Њ) 0.76 Completeness (%) 98.5 (97.9)b Rmerge (%) 5.5 (22.0) Total reflections 92,865 Unique reflections 35,871 Redundancy 2.6 (2.5) Average I/␴(I) 17 (5) Refinement Statistics and Model Quality Resolution range (A˚ ) 40–1.95 Protein nonhydrogen atoms 4,753 Water molecules 160 Sulphate ions 7 c R factor/Rfree (%) 0.178/0.233 Space group P21 Unit cell (A˚ )aϭ 39.60, b ϭ 94.93, c ϭ 67.56 ␤ϭ94.4Њ Rmsd from ideal geometry Bond lengths (A˚ ) 0.012 Bond angles (Њ) 0.86 Ramachandran plotd Most-favored region 94.2 % Additional allowed region 5.8 % Averaged B factors (A˚ 2) NGB* ABCD Main chain 27 24 25 24 Side chain 32 29 28 29 All water molecules 31 a Outer shell statistics are shown in parentheses. The outer shells are 2.6–2.5 A˚ for the absorption peak and inflection point and 2.25–2.20 A˚ for the remote point.