Journal of Inorganic Biochemistry 103 (2009) 1693–1701

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Journal of Inorganic Biochemistry

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Ligand orientation of human neuroglobin obtained from solution NMR andmolecular dynamics simulation as compared with X-ray crystallography

Jia Xu a,1, Lianzhi Li b,1, Guowei Yin a, Haili Li b, Weihong Du a,*

a Department of Chemistry, Renmin University of China, Beijing 100872, Chinab School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China

a r t i c l e i n f o

Article history:Received 21 May 2009Received in revised form 17 September2009Accepted 21 September 2009Available online 26 September 2009

Keywords:Ferric human neuroglobinLigand orientationSolution NMRMD simulation

0162-0134/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jinorgbio.2009.09.016

* Corresponding author. Tel.: +86 10 6251 2660; faE-mail address: whdu@chem.ruc.edu.cn (W. Du).

1 Contributed equally to this work.

a b s t r a c t

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in manybiological processes such as enzymatic reaction, signal transduction and the mitochondria function. Dif-ferent from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imid-azole of proximal His96(F8) and distal His64(E7) directly bound to the metal ion. In the present work,solution 1H NMR spectroscopy was employed to investigate the electronic structure of heme center ofwild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocketwere assigned. Two heme orientations resulting from a 180� rotation about the a–c-meso axis with apopulation ratio about 2:1 were observed. Then the 1H NMR chemical shifts of the ferriheme methylgroups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle u isconsistent with that from the molecular dynamics simulation but not with those from the crystal data.Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobinreflects the changeability of heme environment for the Ngb family.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Neuroglobin (Ngb) [1,2] is a newly discovered globular hemeprotein widely expressed in vertebral nerve systems, retina andendocrine tissues [3,4]. Ngb can bind oxygen reversibly as hemo-globin (Hb) and myoglobin (Mb) [5,6]. However, its low tissue con-centration suggests that Ngb is not an O2 reservoir or transporter.Although the physiologic role of Ngb is still poorly understood[4,7], it is commonly received that Ngb is involved in the activationof a neuroprotective mechanism against hypoxiaischemia [8–12].Besides, many roles of Ngb have also been suggested, such as scav-enging of reactive nitrogen and oxygen species, signal transductionand regulation of apoptotic pathways [4,13–17]. Recent studiesshow that Ngb is associated with the mitochondria function andsuggested the potential therapeutic application in Alzheimer’s dis-ease and neurodegenerative disorders [17–21].

The three dimensional structures of human Ngb (HNgb) andmouse Ngb (MNgb) exhibit a classical three-over-three a-helixsandwich globin fold [22] although they share a low sequenceidentity (<25%) with canoncial Hbs and Mbs [1,23,24]. The ferricand ferrous heme–iron are both hexacoordinated, with histidylimidazole of proximal His96(F8) and distal His64(E7) directly bound

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x: +86 10 6251 6444.

to the metal ion, indicating binding of external ligands (such as O2,CO or NO) can only happen after the rupture of the bond betweeniron and histidyl imidazole of His64(E7) [5,25]. Exogenous ligandsreplace the endogenous ligand His64(E7) in a three-state mecha-nism, of which the rate-limiting step is dissociation of theHis64(E7). Kinetic studies have found a rapid recombination rate(kon(O2) = 300 � 106 M�1 s�1) and a slow dissociation rate(koff(O2) = 0.4 s�1) for both O2 and CO. Spectroscopic characteriza-tion of wild type Ngb and mutagenesis has suggested thatHis64(E7) plays a key role in stabilization of hexacoordination envi-ronment as well as rebinding of exogenous ligands [5,6,26–33].

Besides atypical ligand binding mechanism, Ngb also exhibitsspecial structural heterogeneity and huge internal cavity demon-strated by X-ray crystallography and NMR spectroscopy[22,23,25,34,35]. The binding of exogenous ligand induces confor-mational changes, including a sliding motion of the heme, reposi-tioning of the F-helix and loops of CD and EF, as well asreshaping of inner cavity [23]. Analyzing the ligand orientation ofNgb by EPR reveals that the imidazole planes are no longer parallel,different from those bis-histidyl coordination systems in plant glo-bins [36–39]. Furthermore, the ligand orientations obtained fromsolution NMR data for MNgb display changeable heme center fromthat in the crystal state [40].

To investigate the electronic structure of heme center andligand orientation in solution at ambient temperature for the wildtype ferric HNgb (WT met-HNgb), 1H NMR spectroscopy was

1694 J. Xu et al. / Journal of Inorganic Biochemistry 103 (2009) 1693–1701

utilized in the present work. The 1H chemical shifts of heme andnearby key residues were assigned. And two heme orientationsarising from a 180� rotation about the a–c-meso axis with a pop-ulation ratio about 2:1 in solution were observed. Moreover, theaxial histidine imidazole plane orientations for both isomers ofmet-HNgb predicted upon the heme methyl resonances were com-pared with those of related proteins, such as MNgb and Cytoglobin(Cgb) from NMR and X-ray data. Furthermore, molecular dynamics(MD) simulation was also used to investigate the solution confor-mation of met-HNgb, especially the axial ligand orientations.

2. Materials and methods

2.1. Sample preparation

The WT met-HNgb was expressed and purified as described indetail previously [41]. No DTT was added to reduce the disulfide-bridge. Two samples of met-HNgb (one in 90% 1H2O and 10%2H2O, and another in 99.98% 2H2O), dissolved in 100 mM potas-sium phosphate buffer at pH 7.5, were prepared for the 1H NMRstudy. The final protein concentration of each sample was about2 mM. The pH (not compensated for 2H activity) of the resultingsolutions was adjusted by NaO2H or 2HCl solution.

2.2. NMR spectroscopy

1H NMR spectra in 1H2O and 2H2O were collected on a BrukerAVANCE 600 spectrometer operating at 600 MHz over the temper-ature range 5–35 �C, unless otherwise stated, at a repetition rate of1 s�1 with presaturation of the solvent signal. The proton chemicalshifts were indirectly referenced to 2,20-dimethyl-2-silapentane-5-sulfonate (DSS) through the water resonance calibrated at eachtemperature. 1D 1H NMR spectra were recorded as references,and WEFT [42] pulse program was used to monitor broad, fast re-laxed proton resonances. Standard inversion-recovery experimentwas employed to determine non-selective T1 values for resolvedand fast relaxed resonance. 1D NOE difference experiments werecarried out to observe the nuclear Overhauser enhancements

Fig. 1. 600 MHz 1H NMR spectra of met-HNgb at 303 K, pH 7.5, 100 mM phosphate bufftrace in 2H2O; (C) WEFT spectrum (relaxation delay 20 ms, repetition rate 10 s�1) in 1H2Ogiven superscripts A and B for two isomers, and residues are labeled by position numbe

between selected proton (i.e. 8-CH3 of isomer B or 5-CH3 of isomerA) and other protons.

NOESY [43] and TOCSY [44] spectra were used to identify dipo-lar and scalar connectivities, all using 512 t1 blocks and 4096 t2points. NOESY spectra were collected in 1H2O and 2H2O with a60 ms mixing time. Spectral windows were 42 kHz at 20 and25 �C, and 24 kHz at 25 and 30 �C. TOCSY spectra were recordedat 23 and 28 �C, respectively, with a 40 ms mixing time and band-width of 18 kHz. The 2D data set were processed using Bruker Top-spin software. NOESY spectra were processed by a 30�-shifted-sine-squared-bell apodization in both dimensions and zero-fillingto 4096 � 2048 data points prior to Fourier transformation.

2.3. Molecular dynamics simulation

The 1.95-Å resolution crystal structure of HNgb (PDB code:1OJ6) were taken as the initial structures of HNgb. Chain C, whoseheme orientation was the one like Mb, was chosen to representminor isomer A. For the other three chains, their RMSDs to theaverage of structures are very low (0.47, 0.65, 0.84 Å for chains A,B and D, respectively). Therefore, all the three chains could be usedto represent major isomer B in the MD run. In this work, chain Awhich mostly resembled the average structure was chosen. Pro-tons were added to the crystal coordinates using Sybyl V. 7.3 (Tri-pos Inc., St. Louis, MO, USA) [45] on a Linux platform.

The MD simulations were performed in details as reported [46],with the sander module of Amber 9 package (Scripps, CA, USA) [47]using a standard parm94 force field [48]. The protein was solvatedin a truncated octahedral box of water containing �6700 TIP3Pwater molecules. Moderate Langevein dynamics [49,50] wereadopted for the whole system with a collision frequency ofc = 1 ps�1 to enhance the efficiency of conformational samplingin a solvated state. The nonbonded interaction cutoff value was10.0 Å. The SHAKE algorithm [51], with a tolerance of 10�6, wasturned onto fix all bond lengths involving hydrogen atoms duringthe MD run. The time-step was constant at 2 fs. A 25 ns productionof the MD simulation was then implemented under periodicalboundary conditions in an NTP ensemble at 300 K and 1 atm, usingBerendsen temperature coupling and isotropic molecule-based

er: (A) relaxed (repetition rate 1 s�1) reference trace in 1H2O; (B) relaxed, referencewhich allows detection of strongly relaxed sign at 16.50 ppm. Heme resonances arers and protons.

Table 11H NMR spectral parameters for heme and two histidine ligands signals in met-HNgb.Chemical shifts in ppm are referenced to DSS in 1H2O 100 mM phosphate, pH 7.5,303 K. Non-selective T1 in ms, in parentheses for resolved resonances.

Residue Proton A B

Heme 1-CH3 22.76 (101) 7.03-CH3 17.815-CH3 34.17 13.928-CH3 10.75 35.032-Ha 17.642-Hbc �3.33 �0.702-Hbt �2.62 0.374-Ha �1.33 15.7 (72)4-Hbc �0.90 �5.51 (151)4-Hbt �2.15 �4.116-Ha 16.246-Ha0 2.19 �2.236-Hb 1.89 �0.996-Hb0 0.12 �1.377-Ha 16.307-Ha0 �2.37 8.457-Hb �1.82 1.777-Hb0 �1.33 �0.03

His96(F8) NqH 11.27 11.68CaH 7.17 7.89CbH 11.27 11.43Cb0H 8.17 8.7

His64(E7) NqH 10.15 10.30CaH 7.76 8.16CbH 14.63 13.87 (43)Cb0H 8.81 9.31

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scaling [52]. Ptraj module of the AMBER 9 package was used toanalyze the production of MD runs.

3. Results

3.1. Assignment of heme

The 1H NMR spectra of met-HNgb is shown in Fig. 1. Two sets ofresonances, given superscripts ‘‘A” and ‘‘B”, respectively, are ob-served in the spectra, corresponding to isomers A and B of met-HNgb. Based on the integrations of HB

i and HAi peaks, two isomers

exhibit a proportion ratio of �2:1, just like that in mouse neuroglo-bin [35]. The minor isomer A displays the same heme orientationas in sperm whale Mb, where the side chain of residue Phe42(CD1)appears to close to the heme 5-methyl [53,54]. For major isomer B,the heme orientation is rotated by 180� with respect to the a–c-meso axis (Fig. 2).

The methyl proton resonances in the low-field region were eas-ily resolved by their distinct hyperfine shift and temperature gra-dient. A Curie plot of heme methyl chemical shifts versus inverseof absolute temperature exhibits good linear correlation and veri-fies heme methyl assignments (see Supporting materials Fig. 1s).Moreover, the variable temperature studies were helpful to iden-tify scalar/dipolar connectivities and other hyperfine shifted, re-laxed resonances [55].

The heme protons for each isomer are assigned unambiguouslyfollowing standard 2D NMR assignments protocol [56]. The abso-lute orientation for heme is established on the basis of NOE con-nectivities of heme methyls with assigned heme pocket residues(Fig. 2). For the major isomer B, Phe42(CD1) has a dipolar contactwith 8-CH3. The 4-vinyl group located by TOCSY spectrum exhibitsignificant hyperfine shifts and NOE cross-peaks to the 3-CH3, thus3-CH3, 4-vinyl, 5-CH3 and 6-propionate are assigned by sequentialNOE connectivities. NOEs between the low -field resolved 8-methyl and hyperfine shifted protons identify the 7-propionateand then 1-CH3. For the minor A isomer, Phe42(CD1) has a dipolarcontact with 5-CH3. NOE cross-peaks of 1-CH3 to a vinyl group and8-CH3 to a propionate group identify 2-vinyl and 7-propionate. The6-propionate is assigned by its cross peak to 5-CH3. Most of theheme protons were assigned and the chemical shifts values arelisted in Table 1. The relative hyperfine shifts of heme protonsare very similar to that found in MNgb [35]. Non-selective T1 val-ues for predominantly paramagnetically influenced protons are gi-ven in parentheses.

3.2. Assignments of key residues

Two sets of hyperfine shifted NHCHCH2 spin systems for bothisomers are partially resolved in TOCSY and NOESY spectra. They

Fig. 2. Schematic representation of the heme pocket structure of met-HNgb with face-(vinyl), and P (propionate). (A) Isomer A, the same heme orientation as in sperm whale M

are symptomatic for the backbone protons of two ligated histi-dines, His96(F8) and His64(E7). His96(F8) is further identified as part(AMXi+3) of a helical fragment Glyi-Xi+1-Xi+2-AMXi+3. The exchange-able amide proton and non-exchangeable CbH of His96(F8) havevery close chemical shifts, but they could be distinguished by theH–D exchange 2D NOESY spectrum and the better NiNi+1 NOE con-nection of F-helix. A fast relaxed resonance at 16.50 ppm was ob-served to be exchangeable and have very short T1 (�20 ms), butmore evidences were needed to assign it as NdH of His96. Anotherligated histidine, identified as His64(E7), exhibits a typical alphahelical ai-Ni+3, ai-bi+3 NOE contact with the aliphatic AMX spin sys-tem of an aromatic residue which is assigned as Phe61(E4).

Among the TOCSY connections, a set of up-field hyperfineshifted resonances identified as AM(X3)(Y3) spin system, whichexhibits NOE cross-peaks to 8-CH3 and 1-CH3, is identified asVal101(FG5) for major isomer B. While the near set, exhibitingNOE contacts to 4-vinyl and 5-CH3, is considered as Val101(FG5)for minor isomer A. NOE connections to the side chain ofVal101(FG5) and 3-CH3 observed in the aromatic spectrum windowuniquely identify residue Phe106(G5) for isomer B. In isomer A,

on view from the proximal side. The heme substituents are labeled M (methyl), Vb. (B) Isomer B, the heme orientation 180� rotated with respect to the a–c-meso axis.

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residue Phe106(G5) has visible connections to 2-vinyl instead of 3-CH3. Furthermore, in the aromatic spectrum window, conservedresidue Phe42(CD1) is uniquely identified since its ring protons haveNOE cross-peaks with the heme methyl groups, 8-CH3 and 1-CH3

for isomer B, and 5-CH3 for isomer A, respectively. Most of NOE con-nections have been displayed in Fig. 2 by bidirectional arrows.

Additional key residues such as Gly93(F5), Arg94(F6), Lys95(F7)and Arg97(F9) are identified as part of a-helix by Ni-Ni+1 and ai-Ni+1 as well as some bi-Ni+1 contacts observed in NOESY spectrum(Fig. 3). Trp148(H23) is identified by the NOE contacts withGly93(F5) and His96(F8). All assigned chemical shifts for key resi-dues are listed in Table 2.

The key residues on active site and their NOE contacts producean unambiguous figure between heme and its surroundings for iso-mers A and B (Fig. 2). Heme rotational isomerism indicates unusualheme cavity property of Ngb and implies its special biological func-tion though still unknown. The assigned chemical shifts of hememethyls thus can be utilized to further exploration of axial ligandorientations unquestionably.

Fig. 3. Portions of the NOESY spectrum of met-HNgb in 1H2O at 298 K that illustrates theand part of F-helix connectivities.

3.3. Orientation of the axial histidine ligands

The orientation angle is defined and measured as shown inFig. 4. It is the angle between axial histidine imidazole plane andgeometrical x-axis (N(II)–Fe–N(IV)) of heme plane. The reportedangle is an average angle of those from His96(F8) and His64(E7).Using NMR data, ligand orientation can be predicted by Shift Pat-tern [57], a program visualizing the relationship between hememethyl chemical shifts and ligand orientation of hemoprotein.The contact contribution was estimated using Hückel techniqueswhile pseduocontact contribution was estimated by values ob-tained from model hemes and counter-rotation of the g-tensorwith rotation of the axial ligand nodal plane. Shift Pattern has beenproven to be reliable in analyzing kinds of hemoproteins[40,57,58]. It is easily used according to the order of heme methylchemical shifts and the relative spacing between each two methylchemical shifts.

The order of heme methyl resonances is different for the twoisomers. For isomer B of met-HNgb, the order is 8 > 3 > 5 > 1, since

dipolar contacts between active site residues, such as His64 to Phe61, Gly93 to Trp148,

Table 21H NMR spectral parameters for strongly dipolar shifted active site residues in met-HNgb.

Residue Proton A B

Phe42(CD1) CdHS 7.63 7.55CeHS 8.35 8.27

Phe61(E4) CaH 5.92 6.02CbHS 4.03, 3.03 4.10, 3.21CdHS 7.83 7.90CeHS 6.64 6.72

Leu92(F4) NH 8.77CaH 5.25

Gly93(F5) NH 10.34 10.24CaH 6.07 5.94Ca0H 6.46 6.42

Arg94(F6) NH 9.25 9.36CaH 4.56

Lys95(F7) NH 9.56CaH 4.97

Arg97(F9) NH 10.37Val101(FG5) CaH 3.15 3.29

CbH 2.26 2.50CcH 0.2 �0.04CcH �1.73 �1.36

Phe106(G5) CdHS 8.54 8.66CeHS 7.87 7.82CfH 11.53

Trp148(H23) Ce3H 7.38 7.34Cg2H 8.02 8.05

Fig. 4. Schematic representation of the heme axis system and ligand orientationangle. The U angle defines the direction of the average histidine ring plane(represented in short, thick lines).

J. Xu et al. / Journal of Inorganic Biochemistry 103 (2009) 1693–1701 1697

8-methyl chemical shift (35.03 ppm) is the biggest and 1-methylchemical shift is the smallest (7.0 ppm). Based upon the orderand relative spacing (the relative difference between two methylchemical shifts), the average ligand orientation angle u is about77.5�, using the x-axis as an angle of 0�. For isomer A, the orderis 5 > 1 > 8 > 3. Although the 3-CH3 resonance is not identified,and the chemical shifts of 5-CH3, 1-CH3 and 8-CH3 are almostequally spaced, considering the reverse heme orientation of iso-mers A and B, the corresponding angle u should be at 12.5�(Fig. 5). In order to make comparisons, the corresponding anglesfrom crystal and solution structures for HNgb (crystal), MNgb

(crystal) and Cgb (solution) are also predicted by the same method.All of the obtained angles are shown in Table 3. Further elucidationis described below in discussions.

3.4. Molecular dynamics simulation

MD simulation is used to analyze the orientation of the axialhistidine ligands in view of classical Newtonian mechanism andcompare with the result obtained from NMR data. The method re-flects the steric effects within the protein primarily other than theelectronic effects of the His-Fe bond. After 25 ns MD run at 300 K,the molecular modeling systems of isomers A and B have been en-ergy minimized and reached to equilibrium. The final structurematches the initial structure very well (see Supporting materialsFig. 2s for isomers A and B). The calculated RMSD values for back-bones of isomers A and B are 1.79 and 1.26 Å, respectively.

No water molecule was found to diffuse in or out of the hemecenter, but some around the surface of EF and CD loop regions,which implied possible exogenous ligand pathway and hence im-pacted on the ligand orientation angle. The dihedral angles be-tween axial histidine imidazole planes (His64 or His96) and the x-axis are analyzed by the trajectory files, respectively. The finalaverage angles were from the average of His96 and His64. The axialligand orientation for isomers A and B are obtained and shown inFig. 6. The mean angles of ligand orientation through the 25 nsMD run are 11.8� for isomer A and 78.1� for isomer B, which arequite consistent with that obtained by NMR data.

4. Discussions

The 1H NMR chemical shifts of heme and nearby key residueshave been assigned for the WT met-HNgb. Two sets of resonancesare identified as two different heme orientation isomers A and B.The population ratio of isomers A and B is about 1:2 in solution,which is very similar with WT-MNgb [22,35]. The major isomer Bexhibits a heme orientation rotated 180� about the a, c-meso axis,different from Mb and Cgb [59]. Heme disorder of Ngb reveals itshuge heme cavity property, hexacoordination feature and its un-ique ligand pathway, further special biological functions.

4.1. Properties of axial ligand orientation in met-HNgb

It has been proven that the imidazole plane angle of the axialligand could be predicted by the order and spacing of heme methylresonances due to the effect of axial ligand nodal plane orientationon the contact and pseudocontact shifts, such systems includes Hb,Mb, cytochrome c, etc. [40,58,60–62]. In the present study, the or-der and spacing of heme methyl resonances are obtained from the1H NMR spectra of met-HNgb. And Shift Pattern, a program visual-izing the relative chemical shift of heme methyl groups as a func-tion of angles, is used to plot the axial ligand orientation of met-HNgb as shown in Fig. 5. Angles derived from X-ray structureand MD simulation are also shown in the same figure. The numer-ical values for the chemical shifts are omitted to make this plotstand for general situation and be less affected by other ligands.From heme methyl chemical shifts of met-HNgb, the average li-gand plane angle u for isomer A is 12.5�, and for the opposite hemeorientated isomer B, the angle u is 77.5�. Usually when the heme isturned over, the 6- and 7-propionates simply exchange positionthen dip into the aqueous solution or interact with other chargedresidues. Therefore, the angles for A and B heme orientation is ex-pected to be symmetrical around the a, c-meso axis.

The average imidazole plane angles of axial ligands (His96 andHis64) to x-axis of heme determined from protein X-ray structuresare compared with those obtained from NMR and molecular

Fig. 5. Relative heme methyl shifts (dotted line: M5, square: M1, cycle: M8, solid line: M3) as a function of axial imidazole plane angle u, measured from the x-axis (throughNII–Fe–NIV in the direction of NI–Fe–NIII) for isomers A and B. The average histidine orientations determined by X-ray, MD and NMR are shown in dotted line, dashed line andsolid line, respectively.

Table 3The average ligand imidazole plane angle u obtained from various proteins.

Isomer A (�) Isomer B (�)

Solution structuresHNgb 12.5 77.5MNgba 5.5 84.5Cgb 11.5

Molecular dynamics simulationsHNgb (1OJ6) 11.8 78.1

Crystal structuresHNgb (1OJ6) 5.7 83.6MNgb (1Q1F) 8.8 81.1Cgb (1UTO) 6.3

a From Ref. [40].

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dynamics simulation (Table 3). For WT-HNgb (PDB code: 1OJ6),three (Chains A, B and D) of the four molecules are of same hemeorientation like isomer B, their value of average ligand orientationangle is 83.6�. And for the only molecule chain C, which is of sameheme orientation like isomer A, the orientation angle is 5.7�. Com-pared with crystal structures, we could speculate that when dis-solved in solution, the average ligand orientation of the majorisomer B slightly rotates about 6� clockwise (from 83.6� to 77.5�,see Fig. 4 for u definition), while the average ligand orientationof isomer A also rotates about 6� counterclockwise, as a result ofheme rotating 180� about the a, c-meso axis. The existed differ-ence between ligand angles seen in NMR spectroscopy and X-raycrystallography might be not surprising, considering the huge,open heme cavity, the potential effects of solvation than in crystalstate, and that of disulfide-bridge formation on the whole proteinconformations.

To further investigate the properties of ligand orientation insolution, we carried out MD simulations for the hexacoordinationsystems of two HNgb isomers. After energy minimization and rel-

atively long-time equilibrium, conformations of the protein areoptimized and the simulated systems are credible to better presentreal states in solution. Unexpectedly, the ligand orientation ana-lyzed from the trajectories of MD simulation exhibits high coinci-dence with that from the chemical shifts of heme methyl groups.

The ligand orientation angle was calculated from the average ofHis64 and His96, so even if the average orientation angle did notchange obviously, the angle difference for one of them might bemuch distinct. The heme disorder reflects huge heme cavity ofHNgb, and the axial ligand orientation angles are related to thehexacoordination feature, the exogenous ligand pathway, and theloop region flexibility, therefore they implies the possible enzymereaction and signal transduction of Ngb functions.

4.2. Ligand orientation comparisons of HNgb, MNgb and Cgb

As mentioned above, obvious differences can be observed in theligand orientation when comparing HNgb crystal structure withthat in solution. The ligand orientation rotates clockwise for isomerB and counterclockwise for isomer A. Similar ligand orientation dif-ference can also be observed in MNgb and Cgb, respectively (Table3). Available X-ray structure of Cgb [63] (PDB code: 1UTO) exhibitsprotein conformation similar to isomer A of HNgb and the pre-dicted angle from X-ray is 6.3�. Since the methyl groups resonanceorder is 5 > 1 > 8 > 3 for Cgb [59], moreover, 1-CH3, 8-CH3 and 3-CH3 are almost equally spaced, an average ligand plane angle forCgb is predicted as 11.5�. So the average ligand orientation fromcrystal to solution rotates about 5� counterclockwise. AvailableX-ray structure of MNgb [23] (PDB code: 1Q1F) exhibits proteinconformation similar to isomers A and B of HNgb. And the pre-dicted angle from X-ray is 8.8� to 81.1� for isomer A and B, respec-tively. Furthermore, the reported average ligand orientation anglefor WT met-MNgb in solution was predicted using Shift Patternprogram, resulting 5.5� for isomer A and 84.5� for isomer B [40].

Fig. 6. A plot of the average angles of axial ligand orientation for both A and B isomers calculated from the MD trajectories: (A) for isomer A and (B) for isomer B.

J. Xu et al. / Journal of Inorganic Biochemistry 103 (2009) 1693–1701 1699

Hence, the ligand orientation of MNgb from crystal to solution ro-tates approximately 4� counterclockwise for isomer B and clock-wise for isomer A.

It is quite strange that HNgb and MNgb show different ligandorientation rotation from crystal to solution. For isomer B of met-HNgb, the ligand orientation angles seen in crystal and solutionare 83.6� and 77.5�, respectively, a clockwise rotation from crystalto solution. While for isomer B of met-MNgb, the ligand orientationangles seen in crystal and solution are 81.1� and 84.5�, respectively,a counterclockwise rotation from crystal to solution. The similarsituation is also found for isomer A of met-HNgb and met-MNgb(Table 3). To figure out the problem, Ca-trace structure of HNgb(Chain B in PDB 1OJ6) is overlaid with that of MNgb (PDB code:1Q1F). The comparison indicates that the two structures are rathersimilar to each other, exhibiting a relative low, 1.40-Å RMSD value.But an interesting thing is, the positive charged residue Lys95(F7)lays near the electron-rich 7-propionate group with a distance

(measured from Nf atom of Lys95(F7) side chain to carboxyl O atomof 7-propionate) of 4.32 Å in MNgb and 2.60 Å in HNgb (Fig. 7). As aproximal residue, Lys95(F7) is very close to the fifth ligandHis96(F8), and the strong interaction between itself and 7-propio-nate might induce the clockwise rotation of axial ligand in HNgb.Note that the residue Lys95 in HNgb lies almost on the opposite po-sition than in MNgb, it is not surprising why the average ligand ori-entation angle tends to rotate in the reverse direction for the twoNgbs.

1H NMR spectroscopy provides a simply way to confirm the het-erogeneity of met-HNgb. And the Shift Pattern method successfullypredicts the ligand orientation of hemoprotein. In combinationwith the molecular dynamics simulation, the 1H NMR data revealthe solution structural information on the heme center of met-HNgb that is not in agreement with previous crystal structure ofneuroglobin. The difference of ligand orientation between HNgband MNgb reflects the changeability of heme environment for

Fig. 7. The different orientation of proximal residue Lys95(F7) side chain in HNgb(black) and MNgb (gray). This positive charged residue lies near the electron-rich 7-propionate group with a distance of 4.32 Å in MNgb and 2.60 Å in HNgb (measuredfrom Nf atom of Lys95(F7) side chain to carboxyl O atom of 7-propionate). Theirinteraction might have a potent influence on ligand orientation.

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the Ngb family. These provide more information on Ngb structure-biological function implication.

5. Abbreviations

Ngb neuroglobinmet-HNgb ferric human neuroglobinMb myoglobinHb hemoglobinWT wild typeMNgb mouse neuroglobinCgb cytoglobinMD molecular dynamicsDTT dithiothreitolDSS 2,2-dimethyl-2-silapentane-5-sulfonateWEFT water-eliminated Fourier transformNMR nuclear magnetic resonanceNOE nuclear Overhauser effect2D NOESY two dimensional nuclear Overhauser effect spectroscopy2D TOCSY two dimensional total correlation spectroscopyRMSD root mean square deviation

Acknowledgements

We thank Professor T. Burmester for providing the gene of hu-man neuroglobin friendly and Professor F.A. Walker for her kindlyhelps on Shift Pattern. This work was supported by the NationalBasic Research Program of China (2004CB719900), the NationalNatural Science Foundation of China (20471025) and the Key Pro-ject of the Ministry of Education of China (108121).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jinorgbio.2009.09.016.

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