Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M503814200 on May 24, 2005

J. Biol. Chem., Vol. 280, Issue 29, 27222-27229, July 22, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/29/27222    most recent
M503814200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de Sanctis, D.
Right arrow Articles by Bolognesi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Sanctis, D.
Right arrow Articles by Bolognesi, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Bishistidyl Heme Hexacoordination, a Key Structural Property in Drosophila melanogaster Hemoglobin*

Daniele de Sanctis,a Sylvia Dewilde,bc Clemens Vonrhein,d Alessandra Pesce,a Luc Moens,b Paolo Ascenzi,ef Thomas Hankeln,g Thorsten Burmester,h Marco Ponassi,i Marco Nardini,j and Martino Bolognesijk

From the aDepartment of Physics, National Institute for the Physics of Matter (NFM), and Centre for Excellence in Biomedical Research, University of Genova, Genova I-16146, Italy, the bDepartment of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp B-2610, Belgium, dGlobal Phasing, Ltd., Sheraton House, Castle Park, Cambridge CB3 0AX, United Kingdom, the eDepartment of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, Roma I-00146, Italy, the fNational Institute for Infectious Diseases Lazzaro Spallanzani, Via Portuense 292, Roma I-00149, Italy, the gInstitute of Molecular Genetics, Johannes Gutenberg University of Mainz, Becherweg 32, Mainz D-55099, Germany, the hInstitute of Zoology, Johannes Gutenberg University of Mainz, Müllerweg 6, Mainz D-55099, Germany, the iNational Cancer Institute Genova, Structural Biology Unit, Largo Rosanna Benzi 10, Genova I-16132, Italy, and the jDepartment of Biomolecular Sciences and Biotechnology, INFM, University of Milano, Via Celoria 26, Milano I-20131, Italy

Received for publication, April 8, 2005 , and in revised form, May 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hemoglobins at high concentration have been isolated long ago from some insect larvae living in hypoxic environments. Conversely, a monomeric hemoglobin has been discovered recently in the fruit fly Drosophila melanogaster as intracellular protein expressed both in larvae and in the adult fly. Such a finding indicates that the oxygen supply in insects may be more complex than previously thought, relying not only on O2 diffusion through the tubular tracheal system, but also on carrier-mediated transport and storage. We present here the crystal structure of recombinant D. melanogaster hemoglobin at 1.20 Å resolution. Spectroscopic data show that the protein displays a hexacoordinated heme, whose axial ligands are the proximal and distal His residues. Such bis-His ligation of the heme has sizable effects on the protein local structure. Three protein matrix cavities, comparable in size but not in topological locations with those of sperm whale myoglobin, are spread through the protein matrix; one of these can host a xenon atom. Additionally, D. melanogaster hemoglobin binds one molecule of 3-(cyclohexylamino)propanesulfonic acid (CAPS) buffer at a surface pocket, next to the EF hinge. Despite the high resolution achieved, no sequence/structure features specifically supporting the heme hexa- to pentacoordination transition required for diatomic ligand binding could be recognized.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insects inhale oxygen and exhale carbon dioxide with the aid of the tracheal system, a tubular structure that connects their inner organs to the air. The trachea efficiently supports the passive diffusion of oxygen to the metabolically active tissues. Therefore, the presence of specific respiratory hemolymph proteins for the transport of oxygen has been regarded as unnecessary in this taxon (1). Nevertheless, a few insects possess either of two types of oxygen carriers, hemoglobins (Hbs)1 or hemocyanins (Hcs) (2-4).

A functional Hc has been identified so far only in the nymphs and adults of the stonefly Perla marginata (Plecoptera) (5). This Hc is composed of two distinct subunits and displays cooperative oxygen binding (n ~ 2) with moderately oxygen affinity (P50 ~ 8 torr). No evidence has so far been found for the presence of Hc in the more evolutionarily advanced neopterin insects, suggesting that this type of respiratory protein was later lost in insect evolution (2-4).

Hbs at high concentrations have been found in a few insects that live, at least temporarily, in a hypoxic environment, such as the aquatic larvae of chironomid midges (6), some aquatic backswimmers (7), and the larvae of the horse botfly Gasterophilus intestinalis (8). These species harbor single chain Hbs (~150 amino acids) in millimolar concentrations which either transport oxygen in the hemolymph (midges) or store oxygen in specialized tissues (backswimmers and botfly) (3, 9). Twelve different Hb polypeptides and more than 30 Hb genes have been identified in Chironomus (10-12). All Chironomus Hbs investigated so far have similar oxygen binding properties, are characterized by a myoglobin (Mb)-like oxygen affinity (P50 = 0.6-1.5 torr, at pH 7.0 and 20 °C), are not cooperative heme-proteins, and most of them show an alkaline Bohr effect (13, 14). The three-dimensional structure of Chironomus thummi thummi Hb III, displaying 20% amino acid identity to sperm whale Mb, matches closely the standard globin fold (15-18). These Hbs enable the Chironomus larvae to tolerate nearly anoxic environments (7) and may help explain the spread of more than chironomid species throughout the world (3, 4).

Recently, a monomeric Hb of 153 amino acids (17 kDa) has been discovered in the fruit fly Drosophila melanogaster (DmHb) (19, 20). It is an intracellular protein that is expressed at low concentration in the tracheal system and in the fat body of embryonic, larval, and adult flies. DmHb is produced by four transcripts with identical coding regions but different 5'-exons, which are generated by two distinct promoters of the dmeglob1 gene. Amino acid comparisons show significant sequence similarity with the Hbs from the Chironomidae, whereas the highest similarity score was found with the Hb of G. intestinalis (GiHb; 39% amino acid identity; Fig. 1A). Recombinant DmHb displays a typical hexacoordinated heme absorption spectrum both in the ferric form and in the deoxygenated ferrous form, binding oxygen noncooperatively with an affinity of ~ 0.12 torr (20).



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 1.
A, structure-based amino acid sequence alignments for DmHb, sperm whale Mb, reference insect Hbs, and hexacoordinated Hbs of known three-dimensional structure. SwMb, sperm whale Mb. DmHb 6C is shown in blue. CttHb, C. thummi thummi Hb III; DrHb, Danio rerio Hb (6C); RcHb, rice Hb (6C). 6C indicates known hexacoordinate Hbs. Amino acid sequences are from www.expasy.org/sprot. The topological positions (purple, above the sequences) refer to the classical globin fold, as defined on sperm whale Mb; DmHb sequence numbering is reported in green below the sequences. Residues relevant for the discussion and conserved in the globin fold are highlighted in color. B, schematic comparison of the globin fold in DmHb. A stereo view shows the structural overlay of the C{alpha} backbones of DmHb (green trace) and sperm whale Mb (red trace), identifying the chain termini and the different helical regions according to the standard globin fold nomenclature. Note the main structural deviations occurring in the D region, at the EF helical hinge, and a modest shift of the heme group in the two proteins. For reference, the distal (E7) and proximal (F8) His residues of DmHb have been included (drawn with PyMOL; www.pymol.org).

 
Recent sequence data suggest that similar intracellular Hbs exist in a large variety of insects,2 indicating that the insect oxygen supply can be more complex than previously thought and may partly rely on carrier-mediated oxygen transport and storage (19, 20). The physiological role of insect Hbs expressed at low cellular concentrations is, however, still obscure. Although Hbs, Hcs, and Mbs have long been considered oxygen transport or storage proteins, recent evidence indicates that some heme-proteins serve the detoxification of nitric oxide, the regeneration of NAD+ under anaerobic conditions, or act as oxygen sensors (3, 18, 21-32). Such functions are also conceivable for DmHb. In the context of our ongoing studies on the biological role of hexacoordinated Hbs and on the molecular mechanisms regulating heme-ligand binding and Hb action, we report here the three-dimensional structure of DmHb, the product of the dmeglob1 gene, studied by x-ray diffraction analysis at very high resolution (1.20 Å).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DmHb cDNA was cloned into the pET3a expression vector. Using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), the Cys121 residue at position GH1 was mutated to Ser to avoid irreversible protein aggregation during the crystallization stage. The recombinant plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS. Cells were grown at 25 °C in TB medium containing 200 µg/ml ampicillin, 30 µg/ml chloramphenicol, and 1.0 mM {delta}-aminolevulinic acid. Hb expression was induced at A600 = 0.8 by the addition of isopropyl-1-thio-{beta}-D-galactopyranoside to a final concentration of 0.4 mM, and expression was continued overnight. The bacteria were harvested, dissolved in 50 mM Tris-HCl, pH 8.0, 1.0 mM EDTA, 0.5 mM dithiothreitol, and subsequently broken with three freeze-thaw cycles followed by sonication. The debris was removed by two centrifugation steps (10 min at 10,000 x g, and 60 min at 105,000 x g). Further purification was achieved by 40 and 90% (w/v) (NH4)2SO4 precipitation. The 90% (w/v) (NH4)2SO4 pellet, which contains the crude Hb pellet, was dissolved in 50 mM Tris-HCl, pH 8.5, dialyzed overnight, and loaded onto a Fast Flow DEAE-Sepharose (Amersham Biosciences) column equilibrated in the same buffer. After washing of the unbound material, DmHb was eluted with 200 mM NaCl. The DmHb fractions were concentrated by Amicon filtration (PM10) and passed through a Sephacryl S200 column. The final DmHb fractions were pooled, concentrated, and stored at -20 °C until use (20).

DmHb crystals were grown in the sitting drop vapor diffusion setup, against a reservoir solution containing 34% (w/v) polyethylene glycol 4000, 0.2 M MgCl2, 0.05 M CAPS (pH 10-11.5), at 4 °C. The protein stock solution concentration was 72 mg/ml; crystallization droplets were composed of 1.0 µl reservoir and 1.0 µl stock protein solution. Well shaped prismatic crystals of 300 µm x 100 µm x 5 µm size grew in about 1 week. The crystals chosen for x-ray data collection were transferred to the same buffer medium used for growth, containing 40% (w/v) polyethylene glycol 4000 for cryoprotection. X-ray diffraction data, to a maximum resolution of 1.6 Å, were collected at ESRF (Grenoble, France), on beam line ID29, using one frozen crystal (at 100 K) at wavelength {lambda} = 1.7410 Å (Table I), to maximize the contribution of the anomalous signal of the heme iron atom, after measurement of the crystal x-ray fluorescence spectrum. A high resolution data set was subsequently collected on a crystal from the same batch, at 1.20 Å resolution (at {lambda} = 0.9795 Å; Table I). The collected data were reduced and scaled using MOSFLM and SCALA, respectively (33, 34). The structure was solved using a phased molecular replacement approach; initial phases were obtained with the SAD technique, based on the anomalous scattering of the heme iron atom.


View this table:
[in this window]
[in a new window]
  		TABLE I
Summary of the data collection and refinement statistics for DmHb

 
The iron atom site was located in the {lambda} = 1.7410 Å data set using a combination of SHELXD (35) and visual inspection of anomalous difference Patterson maps. The iron site was used to calculate initial phases using SHARP (36), which were improved through density modification with SOLOMON (37). Subsequently, using as search model the lamprey Hb structure (PDB code 2LHB [PDB] ), phased molecular replacement was performed with MOLREP (38). Further density modification cycles performed again with SOLOMON (37) confirmed the correct solution, which was improved using ARP/wARP (39) through atom addition and deletion. The best map then obtained was successfully autotraced by ARP/wARP (39). Initial refinement was performed with BUSTER-TNT (40), including some side chain adjustments and placing of the heme group.

The partial DmHb model (lacking two amino acids in the N-terminal region, and the Leu76-Glu78 loop) was subsequently refined and completed at 1.20 Å resolution (isotropic B-factors) using the program REFMAC (41), to R-factor and R-free values of 15.3 and 16.7%, respectively. Manual model adjustments were performed using the O suite (42). Finally, individual anisotropic atomic B-factors were refined using REFMAC (41) (R-factor 11.0%, R-free 14.8%), and in the last refinement cycles hydrogen atoms were added (R-factor 10.0%, R-free 13.4%; Table I). The final model contains 153 residues, 318 water molecules, two octahedral aquo-Mg complexes, one chloride anion, and one molecule of the CAPS buffer, with ideal stereochemical parameters (43) (Table I). The estimated atomic positional error is 0.053 Å (44).

Subsequently one crystal was treated in a 20-bar xenon gas pressurized cell, for 20 min; diffraction data were then collected at ESRF on beam line ID23, to the maximum resolution of 1.50 Å. The structure of the DmHb·Xe complex was analyzed first through difference Fourier techniques, then refined using the program REFMAC (41). The corresponding final model contains 153 residues, 199 water molecules, two octahedral aquo-Mg complexes, and two xenon atoms (R-factor 15.8%, R-free 19.2%), with ideal stereochemical parameters (43) (Table I). Absorption spectra, collected on dissolved crystals, indicate that DmHb is present as a mixture of ferrous and ferric forms, already in freshly grown crystals. Protein matrix cavities were calculated using the program Surfnet (45) and a 1.4 Å radius probe (Fig. 2). Atomic coordinates and structure factors for DmHb have been deposited with the Protein Data Bank, as data sets 2BK9 [PDB] and R2BK9, respectively (www.rcsb.org/pdb) (46).



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 2.
Location of protein matrix cavities in DmHb. The mono view shows a DmHb 3-on-3 {alpha}-helical fold. Helices are labeled according to their locations within the globin fold. The heme group is shown in red, together with the heme iron coordinated residues His61(E7) (on the distal site) and His96(F8), on the proximal. The location of the protein matrix cavities identified in DmHb is shown by purple mesh and numbered according to text. The protein scaffold has approximately the same orientation as Fig. 1B.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall DmHb Structure—The three-dimensional structure of DmHb (bearing the Cys121(GH1) -> Ser mutation) was solved by phased molecular replacement, with initial phases obtained through the SAD method, based on the anomalous signal of the heme iron atom. Refinement of the crystal structure, including anisotropic B-factor refinement and fixed hydrogen atoms, converged at a conventional R-factor value of 10.0% (R-free 13.4%), at 1.20 Å resolution (Table I).

As implied by the amino acid sequence and supported by the molecular replacement technique adopted in the phasing, the DmHb protein fold matches well the three-over-three {alpha}-helical sandwich fold of classical Hbs and Mbs, where the eight helices building up the globin fold are conventionally labeled A-H, according to their sequential order (47-50). Structural overlay of the DmHb structure on sperm whale Mb (PDB code 1A6N [PDB] ), taken as the parent globin structure, yields a r.m.s. deviation of 2.18 Å (calculated over 134 C{alpha} pairs) (Fig. 1B). The largest deviations occur in the CD-D region, where DmHb displays a scarcely helical D segment, in the N-terminal region of the E-helix, in the following EF hinge, which is three residues longer in DmHb, and in the GH hinge, two residues longer in sperm whale Mb (Fig. 1A). A modest (0.5 Å) shift of the heme group toward the EF interhelical hinge region is observed in sperm whale Mb, relative to DmHb. A comparably high r.m.s. deviation value (2.0 Å, for 75 C{alpha} pairs) is found when the DmHb backbone is compared with that of Hb III from the larvae of the midge C. thummi thummi (PDB code 1ECO [PDB] ). In this case, structural deviations appear to be distributed throughout the protein chain, with a tendency to higher deviations in the F-G-H helical segments. Additionally, the heme group in DmHb appears to be more deeply located within the heme crevice, by about 2.7 Å, relative to C. thummi thummi Hb III, possibly related to 100% heme isomerism observed in the crystal structure of the latter protein (17).

Because the heme group in DmHb displays a bis-His hexacoordination (see below), it is of particular interest to examine the protein structure in relation to other hexacoordinate Hbs recently reported, particularly human cytoglobin (CYGB; PDB code 1UTO [PDB] ), human neuroglobin (NGB; PDB code 1OJ6 [PDB] ), mouse neuroglobin (mNgb; PDB code 1Q1F [PDB] ), Caudina arenicola Hb (PDB code 1HLB [PDB] ), and rice nonsymbiotic Hb (PDB code 1D8U [PDB] ) (29, 51-58). Structural overlay of DmHb on CYGB yields a r.m.s. deviation of 0.98 Å for 110 C{alpha} pairs, thus indicating a very good match to the CYGB C{alpha} trace. As noted above for sperm whale Mb, a two-residue insertion in DmHb EF hinge causes some local structural deviation in the two proteins, which also deviate somewhat at the C-terminal region of the G helix. Overlay of DmHb and NGB structures yields a r.m.s. deviation of 1.31 Å for 85 C{alpha} pairs. The main differences are located in the protein CD region, related to the widely open conformation displayed by the CD heme distal region in NGB. Deviations at the EF hinge are also present, as noted above, related to a three-residue insertion in DmHb. Structural overlay on C. arenicola Hb yields a r.m.s. deviation of 1.76 Å (calculated over 107 C{alpha} pairs). The main differences in comparing the two structures are located in the extended N-terminal region of C. arenicola Hb which has no counterparts in the DmHb structure, in the EF hinge, with a three-residue insertion in DmHb, and in the GH loop, which is two residues longer in C. arenicola Hb. The overlay of DmHb and rice Hb yields a r.m.s. deviation of 2.17 Å, for 83 C{alpha} pairs. Again, the main differences are observed in the CD region, where the rice Hb structure is poorly defined, and in the EF-F regions. It should be noted that the EF hinge region is more extended in rice Hb than in other globins, whereas the F helix is shorter by five residues (52).

Heme Distal and Proximal Sites in DmHb—One of the most striking features of the DmHb overall structure is the bis-His coordination to the heme iron atom, which is observed in the crystal structure in agreement with expectations based on the protein spectral properties (20). The DmHb distal site displays a coordination bond connecting the heme iron atom to the distal His61(E7) NE2 atom, with a bond length of 2.02 Å. The His61(E7) imidazole ring is almost perfectly staggered relative to the heme pyrrole nitrogen atoms, being oriented toward the methinic bridge CHA and CHC atoms of the porphyrin ring. The average heme nitrogen(pyrrole)-iron distance is 2.0 Å; the iron atom appears well within the heme nitrogen(pyrrole) plane, the iron-His61(E7) coordination bond being almost orthogonal to the heme plane (Fig. 3).

The coordination of the distal His61(E7) residue to the heme iron atom results in a shift of the N-terminal region of the E helix of about 4.3 Å (at residue Ala56(E2)) relative to the orientation adopted in sperm whale Mb. Such a shift affects approximately one-third of the E helix, locating it closer to the heme (Fig. 1B). In particular, at the distal His61(E7) residue, the DmHb protein backbone is closer to the heme by about 2.6 Å relative to sperm whale Mb, a structural shift sufficient to support DmHb His61(E7) direct coordination to the iron atom. In keeping with such an observation, a comparable shift of the N-terminal part of the E helix toward the heme, relative to sperm whale Mb, is observed in rice Hb, in CYGB, and in NGB, all being bis-His hexacoordinated globins (29, 52-58).

The heme-coordinated His61(E7) residue is surrounded by apolar distal residues (the nearest neighbors are Leu28(B10), Phe42(CD1), and Ile65(E11)). No polar residues or ordered water molecules are present inside the heme distal pocket, where no protein cavity in the immediate neighborhood of the heme can be recognized when the distal His61(E7) is bound to the heme iron atom. The surface residue Arg64(E10) may be electrostatically coupled to the A heme propionate (5.5 Å distance) and is hydrogen-bonded to surface water molecules. In the orientation displayed in the DmHb crystal structure, the Arg64(E10) guanidinium group is oriented toward the heme distal pocket and His61(E7), to which it is connected by a bridging hydrogen-bonded water molecule (Wat41). In this way, a substantial fraction, although not all, of the surface access to the heme distal site is shielded from solvent. A distal site solvent access region remains partly open between the side chain of Arg57(E3) and the heme D propionate, where two ordered water molecules are located (Wat66 and Wat180). Although such an opening in the protein structure may allow an external diatomic ligand to contact the porphyrin ring marginally, in the observed heme hexacoordinated structure any further access to the heme iron atom is prevented.



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 3.
Heme cavity in DmHb. A stereo view of the final 1.2 Å resolution electron density in the surroundings of the heme group in DmHb shows details of the heme iron atom hexacoordination and of the porphyrin ring. The orientation of the heme cavity is approximately that of Fig. 2. Key residues in the heme cavity have been labeled according to their topological positions (drawn with PyMOL; www.pymol.org).

 



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 4.
A, DmHb molecular surface properties. An electrostatic map of DmHb shows the localization of a positive charge around the heme crevice and in general over the protein surface. The protein molecule is displayed in an orientation comparable with that of the previous figures. The surface charge distribution has been calculated with the program MEAD (75). B, view of the protein surface, coded for electrostatics as in A, showing the marked surface cleft at the EF hinge region where the buffer CAPS molecule (shown as stick model) is bound. The heme group and {alpha}-helical segments are visible under the translucent surface (drawn with PyMOL; www.pymol.org).

 
Concerning the overall protein structure on the heme distal side, it should be further noted that half of the distal E helix displays a localization of positive charges, with four Arg residues (Arg57(E3), Arg59(E5), Arg64(E10), and Arg67(E13)) (Fig. 4A). These surround with a positive electrostatic field the distal His61(E7) residue, namely a site that may be pivotal in the diffusion of ligands to/from the heme and in their stabilization. On the other hand, most of the DmHb molecular surface is characterized by a striking distribution of negative charges (Fig. 4A). In this respect, the negative charge widespread over the protein surface is in keeping with the monomeric state achieved by DmHb in solution (19, 20).

The proximal His96(F8) residue provides a coordination bond of 1.97 Å to the heme iron atom, being staggered relative to the heme pyrrole nitrogen atoms. Furthermore, because the His96(F8) imidazole ring is oriented along the heme methinic CHB-CHD atoms direction, the distal and the proximal His imidazole rings display almost orthogonal (88°) relative azimuthal orientations. The azimuthal orientation of the His96(F8) imidazole ring is further stabilized, as often in globins (18), by a hydrogen bond between the histidyl ND1 atom and the Ile92(F4) carbonyl oxygen atom (2.76 Å). Remarkably, and different from other known globin structures, the last turn of the F helix deviates substantially from a regular {alpha}-helical conformation, likely in relation to the presence of a prolyl residue at the C-terminal end of the helix (Pro98(F10)). The local hydrogen bonding pattern is strongly perturbed, such that the Val94(F6)-Pro98(F10) stretch is best described as a 310 helix. Nevertheless, coordination to the heme iron atom is achieved according to conventional globin schemes (18, 29, 32, 59), the coordination bond being almost orthogonal to the porphyrin plane. The occurrence of Pro residues at this globin topological site is uncommon, being observed in less than 3% of all known globin sequences (3, 50).

Binding of the heme group to the protein is stabilized by more than 50 van der Waals contacts (<4 Å) and by ionic interactions. Residues Arg57(E3) and Arg64(E10), on the heme distal side, are involved in a network of hydrogen-bonded interactions mediated by water molecules, including propionates D and A, respectively. On the proximal heme side the guanidinium of Arg99(FG2) falls at 2.9 Å from the heme propionate D, indicative of a hydrogen-bonded salt link. The porphyrin ring displays a rather planar structure; the heme atoms deviate by 0.22 Å r.m.s. from the average porphyrin plane (propionates have been excluded from the calculation). Furthermore, analysis of heme distortion along normal modes using the program NSD (60) indicates that the contained heme distortions are essentially caused by out-of-plane ruffling, saddling, and doming main modes, with values of 0.32, 0.28, and 0.24 Å, respectively.

In consideration of the high resolution refinement, analysis of the atomic anisotropic B-factors shows essentially an isotropic behavior for the protein atoms building the heme pocket. An interesting deviation from this pattern is displayed by the imidazole rings of His61(E7) and His96(F8). In fact, both their NE2 atoms display nearly spherical isotropy, whereas both their ND1 and CE1 atoms display anisotropic displacements. These observations can be reconciled with the presence of the heme iron coordination bonds, restraining atomic oscillation of the distal/proximal NE2 atoms, on one hand, with a preferential libration of both imidazole rings around the NE2(proximal)-iron-NE2(distal) direction, on the other. Measurement of the DmHb continuous wave EPR spectra are in excellent agreement with these results.3

Protein Matrix Cavities—Core cavities have been previously recognized and mapped in globins (61). This structural property has been related to a functional role as temporary docking stations for small gaseous ligands in sperm whale Mb (27, 62). Protein matrix cavity/tunnel systems located next to the heme highlighted in NGB, mNgb, CYGB, Cerebratulus lacteus mini-Hb, and truncated Hbs, have been related to the different functional properties displayed by these proteins (24, 32, 53-59, 63-65). Indeed, in hexacoordinated Hbs the requirement for structural readjustments at the His(E7) residue, and/or at neighboring residues, or for the heme fine positioning, may take advantage of such an empty space during the transition from a bishistidyl hexacoordinated Hb to a protein bearing an exogenous ligand at the heme distal site.

Inspection of the DmHb structure shows three core cavities (empty volumes of 32, 23, and 23 Å3, respectively), which may provide temporary docking sites for small diatomic ligands (Fig. 2). Both the first and the second cavity are located on the distal side of the heme; the larger (cavity 1) falls at about 13 Å from the heme iron atom, nestled between the B, E, and G helices, being defined by the hydrophobic residues Trp13(A12), Ile65(E11), Ile66(E12), Phe69(E15), Ile113(G12), and Leu117(G-16). Cavity 2 is located further away from the heme, in the direction of the A helix and of the GH hinge region, about 7 Å away from cavity 1. It is separated from cavity 1 by residues Trp13(A12), Pro16(A15), Leu117(G16), and Trp131(H8). Cavity 2 is close to the protein surface, being defined on this side by solvent-exposed residues Thr12(A12), Ser121(G20), and Leu123(-GH1). In the context of DmHb dynamic behavior it is possible that cavities 1 and 2 merge, yielding a single elongated apolar cavity. Contained conformational readjustments on the solvent side of cavity 2 could open it to external ligands. Moreover, cavity 1 is separated from the distal heme pocket by residues Ile65(E11) and Phe69(E15); a modest conformational shift in one of these could then complete a temporary (continuous or discontinuous) access path for small ligands to/from the heme distal cavity. In this context, it should also be noted that Ser121(G20), one of the exposed residues defining the solvent interface of cavity 2, is the residue mutated (from Cys) in the crystallized protein. No evidence of effects of the mutation on the DmHb ligand binding properties, however, has been so far recorded.4

The third cavity is located on the heme proximal side. It is defined by the residues Ile97(F9) and Tyr106(G5), by the C-terminal backbone of the F helix (including His96(F8)), by the backbone of the FG loop, and by the protein C-terminal region. Two water molecules (Wat1 and Wat4), mutually hydrogen-bonded, but also linked to the carbonyl oxygen atoms of Ala93(F5), Val101(G1), Gly150, and to the phenolic oxygen atom of Tyr106(G5), fill cavity 3. Such water molecules fall at 8-9 Å from the heme iron atom (see Fig. 2).

To gain insight on the possible role played by the observed cavities and on their ligand accessibility, one DmHb crystal was treated with pressurized xenon gas, and a diffraction data set was collected at 1.50 Å resolution (see "Experimental Procedures"). The refined structure shows that one xenon atom is bound at cavity 1 (50% occupancy). Moreover, some weak electron density may account for a low occupancy (20%) xenon atom, at about 7 Å from the first site, separated by residue Phe69(E15), which displays a double conformation. Cavity 2, nestled between the A helix and the GH loop, is not reached by the xenon atoms, whereas cavity 3 still displays two bound water molecules.

Solvent Structure and DmHb-bound Species—As mentioned above, one molecule of CAPS was unambiguously identified as a residual electron density peak. The buffer molecule is hosted in a low polarity surface pocket, located between the EF region, the H helix, and the N terminus of the protein. The sulfonate group of CAPS is electrostatically linked to residues Arg57(E3) and Arg64(E10) of a crystal symmetry equivalent molecule and hydrogen-bonded through bridging water molecules (Fig. 4B). The CAPS molecule appears to be kept at this site only by the above packing interactions and by contacts of the cyclohexyl ring with the apolar surroundings provided by the protein, the propanesulfonate tail being solvent-exposed. It is therefore possible that CAPS binds to DmHb only after the crystal contacts are established, partly taking advantage of a preexisting protein surface pocket. It is worth stressing that such a pocket can be achieved in DmHb thanks to a three-residue insertion in the EF hinge region, which results in an F helix particularly elongated at its N terminus, where CAPS binds (Fig. 4B).

While screening for DmHb crystallization conditions it became clear that a divalent ion, such as Mg2+, was a successful crystal growth additive. Analysis of the refined structure shows two hexacoordinated Mg2+ ion complexes at nearby sites on the protein surface, next to the C-terminal region of the protein. In one case the Mg2+ ion is octahedrally coordinated to six water molecules; in the other, one of the six Mg2+ ligands is provided by the carboxylate of Asp147(H24), the others being water molecules. The two Mg2+ complexes appear to play a crystal lattice stabilization role for four symmetry-equivalent DmHb molecules, through interaction with the predominantly negatively charged protein surface.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Binding of a diatomic ligand to any Hb requires first diffusion of the ligand to the heme, a process that may be achieved in many different ways, such as by opening of the distal His(E7) gate or by migration through the protein matrix, based on specific cavity/tunnel systems (24, 27, 29, 32, 53, 54, 56-59, 63-67). In bis-His hexacoordinated Hbs, removal of the heme-coordinated distal His(E7) residue prior to ligand diffusion to the heme is also required (29, 68). Such a process, which can be compared with the removal of the heme-coordinated water molecule or of the hydroxyl ion observed in some ferric heme-proteins (69, 70), substantially slows down the ligand association process, in that it may require extensive conformational readjustment of the distal site structure (32). Speculations about this process have been put forward for vertebrate NGB and CYGB (53, 54, 57), where striking flexibility of the CD-D protein region, or a double conformation for the N-terminal part of the E helix, have been observed. Involvement of these regions in a mechanism facilitating removal of the heme distal His prior to ligand coordination to the heme, however, is not yet proved. It is nevertheless interesting to notice also that in hexacoordinated nonsymbiotic rice Hb, the CD-D region is largely disordered (52). In this respect, a recent crystal structure analysis of mNgb has shown that binding of carbon monoxide to the bis-His hexacoordinated species can be achieved without major conformational changes in the protein backbone, but through a sizable shift of the porphyrin ring within the heme crevice, providing enough room for the diatomic ligand (58).

The present crystallographic approach to DmHb shows that no evident structural signature for hexacoordinated Hbs can be recognized in the protein primary and tertiary structures. In fact, residue identities between penta- and hexacoordinated Hbs are widespread over the whole sequence length, and three-dimensional folds are relatively well conserved. Such an observation suggests that the transition between the different coordination states of the globins may be subtly rooted in their dynamics rather than in explicit sequence/structural features. Other selected globin systems achieve hexacoordination only under specific environmental conditions, such as high pH in Chlamydomonas eugametos truncated Hb (71). An important structural role in DmHb must be played by residues ensuring the achievement of fully staggered azimuthal orientations for the distal and proximal His residues. Such a property may play a central role in promoting coordination of the heme iron atom from the distal site in an easily reversible way, by making the iron atom properly available to the incoming endogenous ligand (i.e. His(E7)).

Many model globin structures, whose heme iron atom is pentacoordinated in the ferrous state, have shown that a change in the oxidation state of the iron atom occurs with a change in coordination of the metal, which may bind a water molecule or an hydroxyl ion in the ferric state. The associated heme distal site reorganization energy may therefore limit the kinetics of heme iron reduction. In keeping with this hypothesis, it has been observed that hexacoordinated Hbs display a faster heme iron reduction kinetics, using dithionite, compared with sperm whale Mb and other globins whose heme iron atom is coordinated to a water molecule in the ferric state. Such an observation would suggest that a bishistidyl heme species would be an evolutionary achievement, gained by specific globins, to cope with the need for a fast reduction process, such as that required in the pseudocatalytic activity associated to the scavenging of nitrogen- and oxygen-reactive species (72). In accord with this suggestion, a close correlation between the heme iron atom coordination state, the autoxidation rate, and the heme reactivity toward O2 has been reported for midge larval Hbs (73).

From an alternative viewpoint, the resemblance of evolutionary origin, expression patterns, and oxygen affinity between related penta- and hexacoordinated Hbs may support the hypothesis of a similar function in oxygen homeostasis. In this case, Hb hexacoordination might be interpreted as a structural feature controlling the kinetics of (excessively fast) oxygen rebinding to the heme (29, 72). Such a feature may not be favorable for oxygen storage proteins that function under acute hypoxic conditions but may guarantee a continuous flow of oxygen to the respiratory chain in normoxia.

The respiratory function of the pentacoordinated Hbs, which accumulate in some insects at high concentrations in the hemolymph (chironomids) or in specialized organs associated with the trachea (botfly), is undisputed (3). The presence of hexacoordinated Hbs in this taxon has only recently emerged (20, 28), but their role remains to be established; DmHb is such a hexacoordinated, intracellular globin. Although the total expression level of DmHb appears to be lower compared with chironomid and botfly globins, DmHb shares striking similarities with the pentacoordinated Hb of the botfly G. intestinalis. Indeed, D. melanogaster and G. intestinalis Hb genes are probably orthologous, i.e. they diverged by speciation (28). Both genes contain two introns, located at the identical positions D7.0 (i.e. between codons 6 and 7 of helix D), and G7.0. Other similarities include the tissue distribution (fat body in D. melanogaster and fat body-associated tracheal cells in G. intestinalis) and total oxygen affinity (0.12 torr in D. melanogaster and 0.15 torr in G. intestinalis). A respiratory function may thus be considered as the likely role for both Hbs. Under this hypothesis, DmHb present in tracheoles and in the terminal cells of the trachea may either serve as an oxygen buffer (to compensate for hypoxia periods), or it may facilitate a permanent oxygen flow from the tracheal space into the surrounding tissues. However, spectroscopic properties and preliminary structural data indicate that G. intestinalis Hb is not a hexacoordinate Hb.5


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 2BK9 and R2BK9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by a grant from the Italian National Research Council Project PS-Functional Genomics, Grant RBAU015B47_002 from the Ministry for University and Scientific Research, Grant Bu956/6-1 from the Deutsche Forschungsgemeinschaft, and a grant from the European Community Project QRTL-2001-01548. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

c Postdoctoral fellow of the Fund for Scientific Research Flanders. Back

k Supported by the Istituto Giannina Gaslini (Genova, Italy) and the Fondazione Compagnia di San Paolo (Torino, Italy). To whom correspondence should be addressed. Tel.: 39-02-5031-4893; Fax: 39-02-5031-4895; E-mail: martino.bolognesi{at}unimi.it.

1 The abbreviations used are: Hb, hemoglobin; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CYGB, human cytoglobin; DmHb, D. melanogaster hemoglobin; GiHb, G. intestinalis Hb; Hc, hemocyanin; Mb, myoglobin; mNgb, mouse neuroglobin; NGB human neuroglobin; r.m.s., root mean square. Back

2 T. Hankeln, unpublished results. Back

3 I. Ioanitescu, S. Van Doorslaer, S. Dewilde, and L. Moens, personal communication. Back

4 I. Moens and S. Dewilde, unpublished results. Back

5 A. Pesce, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Gerarde Bricogne (Global Phasing, Ltd.) for support with some of the key experimental steps in this work. We are grateful for access to the ESRF ID29 beam line.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Willmer, P., Stone, G., and Johnston, I. A. (2000) Environmental Physiology of Animals, Blackwell Press, Oxford
  2. van Holde, K. E., and Miller, K. I. (1995) Hemocyanins Adv. Protein Chem. 47, 1-81
  3. Weber, R., and Vinogradov, S. N. (2001) Physiol. Rev. 81, 569-628[Abstract/Free Full Text]
  4. Burmester, T. (2002) J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 172, 95-107
  5. Hagner-Holler, S., Schoen, A., Erker, W., Marden, J. H., Rupprecht, R., Decker, H., and Burmester, T. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 871-874[Abstract/Free Full Text]
  6. Osmulski, P., and Leyko, W. (1986) Comp. Biochem. Physiol. B 85, 701-722
  7. Wells, R. M. G., Hudson, M. J., and Brittain, T. (1981) J. Comp. Physiol. 142, 515-522
  8. Dewilde, S., Blaxter, M., Van Hauwaert, M. L., Van Houte, K., Pesce, A., Griffon, N., Kiger, L., Marden, M. C., Vermeire, S., Vanfleteren, J., Esmans, E., and Moens, L. (1998) J. Biol. Chem. 273, 32467-32474[Abstract/Free Full Text]
  9. Miller, P. L. (1966) J. Exp. Biol. 44, 529-543[Abstract/Free Full Text]
  10. Green, B. N., Kuchumov, A. R., Hankeln, T., Schmidt, E. R., Bergtrom, G., and Vinogradov, S. N. (1998) Biochim. Biophys. Acta 1383, 143-150[CrossRef][Medline] [Order article via Infotrieve]
  11. Hankeln, T., Friedl, H., Ebersberger, I., Martin, J., and Schmidt, E. R. (1997) Gene (Amst.) 205, 151-160[CrossRef][Medline] [Order article via Infotrieve]
  12. Hankeln, T., Amid, C., Weich, B., Niessing, J., and Schmidt, E. R. (1998) J. Mol. Evol. 46, 589-601[CrossRef][Medline] [Order article via Infotrieve]
  13. La Mar, G. N., Overkamp, M., Sick, H., and Gersonde, K. (1978) Biochemistry 17, 352-361[CrossRef][Medline] [Order article via Infotrieve]
  14. Weber, R. E., Braunitzer, G., and Kleinschmidt, T. (1985) Comp. Biochem. Physiol. B 80, 747-753[CrossRef][Medline] [Order article via Infotrieve]
  15. Huber, R., Epp, O., Steigemann, W., and Formanek, H. (1971) Eur. J. Biochem. 19, 42-50[Medline] [Order article via Infotrieve]
  16. Weber, R. E., Bonaventura, J., Sullivan, B., and Bonaventura, C. (1978) J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 123, 177-184[CrossRef]
  17. Steigemann, W., and Weber, E. (1979) J. Mol. Biol. 127, 309-338[CrossRef][Medline] [Order article via Infotrieve]
  18. Bolognesi, M., Bordo, D., Rizzi, M., Tarricone, C., and Ascenzi, P. (1997) Prog. Biophys. Mol. Biol. 68, 29-68[CrossRef][Medline] [Order article via Infotrieve]
  19. Burmester, T., and Hankeln, T. (1999) Mol. Biol. Evol. 16, 1809-1811[Medline] [Order article via Infotrieve]
  20. Hankeln, T., Jaenicke, V., Kiger, L., Dewilde, S., Ungerechts, G., Schmidt, M., Urban, J., Marden, M. C., Moens, L., and Burmester, T. (2002) J. Biol. Chem. 277, 29012-29017[Abstract/Free Full Text]
  21. Hardison, R. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5675-5679[Abstract/Free Full Text]
  22. Burmester, T., Ebner, B., Weich, B., and Hankeln, T. (2002) Mol. Biol. Evol. 19, 416-421[Abstract/Free Full Text]
  23. McMahon, T. J., Moon, R. E., Luschinger, B. P., Carraway, M. S., Stone, A. E., Stolp, B. W., Gow, A. J., Pawloski, J. R., Watke, P., Singel, D. J., Piantadosi, C. A., and Stamler, J. S. (2002) Nat. Med. 8, 711-717[Medline] [Order article via Infotrieve]
  24. Wittenberg, J. B., Bolognesi, M., Wittenberg, B. A., and Guertin, M. (2002) J. Biol. Chem. 277, 871-874[Free Full Text]
  25. Wainwright, W. G., and Poole, R. (2003) Adv. Microb. Physiol. 47, 255-310[Medline] [Order article via Infotrieve]
  26. Wittenberg, J. B., and Wittenberg, B. A. (2003) J. Exp. Biol. 206, 2011-2020[Abstract/Free Full Text]
  27. Brunori, M., Bourgeois, D., and Vallone, B. (2004) J. Struct. Biol. 147, 223-234[CrossRef][Medline] [Order article via Infotrieve]
  28. Burmester, T., and Hankeln, T. (2004) News Physiol. Sci. 19, 1010-1113
  29. de Sanctis, D., Pesce, A., Nardini, M., Bolognesi, M., Bocedi, A., and Ascenzi, P. (2004) IUBMB Life 56, 643-651[Medline] [Order article via Infotrieve]
  30. Weber, R., and Fago, A. (2004) Respir. Physiol. Neurobiol. 144, 141-159[CrossRef][Medline] [Order article via Infotrieve]
  31. Hankeln, T., Ebner, B., Fuchs, C., Gerlach, F., Haberkamp, M., Laufs, T. L., Roesner, A., Schmidt, M., Weich, B., Wystub, S., Saaler-Reinhardt, S., Reuss, S., Bolognesi, M., de Sanctis, D., Marden, M. C., Kiger, L., Moens, L., Dewilde, S., Nevo, E., Avivi, A., Weber, R. E., Fago, A., and Burmester, T. (2005) J. Inorg. Biochem. 99, 110-119[CrossRef][Medline] [Order article via Infotrieve]
  32. Milani, M., Pesce, A., Nardini, M., Ouellet, H., Ouellet, Y., Dewilde, S., Bocedi, A., Ascenzi, P., Guertin, M., Moens, L., Friedman, J. M., Wittenberg, J. B., and Bolognesi, M. (2005) J. Inorg. Biochem. 99, 97-109[CrossRef][Medline] [Order article via Infotrieve]
  33. Leslie, A. G. M. (2003) MOSFLM User Guide, Mosflm Version 6.2.3, MRC Laboratory of Molecular Biology, Cambridge, UK
  34. Evans, P. R. (1993) Proceedings of the CCP4 Study Weekend, on Data Collection and Processing, pp. 114-122, CLRC Daresbury Laboratory, UK
  35. Schneider, T. R., and Sheldrick, G. M. (2002) Acta Crystallogr. Sect. D 58, 1772-1779[CrossRef][Medline] [Order article via Infotrieve]
  36. La Fortelle, E., and de Bricogne, G. (1997) Methods Enzymol. 276, 472-494[CrossRef]
  37. Abrahams, J. P., and Leslie, A. G. W. (1996) Acta Crystallogr. Sect. D 52, 30-42[CrossRef][Medline] [Order article via Infotrieve]
  38. Vagin, A., and Teplyakov, A. (1997) J. Appl. Crystallogr. 30, 1022-1025[CrossRef]
  39. Perrakis, A., Harkiolaki, M., Wilson, K. S., and Lamzin, V. S. (2001) Acta Crystallogr. Sect. D 57, 1445-1450[CrossRef][Medline] [Order article via Infotrieve]
  40. Blanc, E., Roversi, P., Vonrhein, C., Flensburg, C., Lea, S. M., and Bricogne, G. (2004) Acta Crystallogr. Sect. D 60, 2210-2221[CrossRef][Medline] [Order article via Infotrieve]
  41. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  42. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. D 47, 110-119
  43. Engh, R. A., and Huber, R. (1991) Acta Crystallogr. Sect. A 47, 392-400[CrossRef]
  44. Cruickshank, D. W. (1999) Acta Crystallogr. Sect. D 55, 583-601[CrossRef][Medline] [Order article via Infotrieve]
  45. Laskowsi, R. A. (1995) J. Mol. Graph. 13, 323-330[CrossRef][Medline] [Order article via Infotrieve]
  46. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) Nucleic Acids Res. 28, 235-242[Abstract/Free Full Text]
  47. Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386[CrossRef][Medline] [Order article via Infotrieve]
  48. Bashford, D., Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 199-216[CrossRef][Medline] [Order article via Infotrieve]
  49. Holm, L., and Sander, C. (1993) FEBS Lett. 315, 301-306[CrossRef][Medline] [Order article via Infotrieve]
  50. Moens, L., Vanfleteren, J., Van de Peer, Y., Peeters, K., Kapp, O., Czeluzniak, J., Goodman, M., Blaxter, M., and Vinogradov, S. (1996) Mol. Biol. Evol. 13, 324-333[Abstract]
  51. Mitchell, D. T., Kitto, G. B., and Hackert, M. L. (1995) J. Mol. Biol. 251, 421-431[CrossRef][Medline] [Order article via Infotrieve]
  52. Hargrove, M. S., Brucker, E. A., Stec, B., Sarath, G., Arredondo-Peter, R., Klucas, R. V., Olson, J. S., and Phillips, G. N., Jr. (2000) Structure Fold Des. 8, 1005-1014[Medline] [Order article via Infotrieve]
  53. Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln, T., Burmester, T., and Bolognesi, M. (2003) Structure 11, 1087-1095[Medline] [Order article via Infotrieve]
  54. de Sanctis, D., Dewilde, S., Pesce, A., Moens, L., Ascenzi, P., Hankel, T., Burmester, T., and Bolognesi, M. (2004) J. Mol. Biol. 336, 917-927[CrossRef][Medline] [Order article via Infotrieve]
  55. de Sanctis, D., Dewilde, S., Pesce, A., Moens, L., Ascenzi, P., Hankel, T., Burmester, T., and Bolognesi, M. (2004) Biochem. Biophys. Res. Commun. 316, 1217-1221[CrossRef][Medline] [Order article via Infotrieve]
  56. Sugimoto, H., Makino, M., Sawai, H., Kawada, N., Yoshizato, K., and Shiro, Y. (2004) J. Mol. Biol. 339, 873-885[CrossRef][Medline] [Order article via Infotrieve]
  57. Vallone, B., Nienhaus, K., Brunori, M., and Nienhaus, G. U. (2004) Proteins 56, 85-92[CrossRef][Medline] [Order article via Infotrieve]
  58. Vallone, B., Nienhaus, K., Matthes, A., Brunori, M., and Nienhaus, G. U. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 17351-17356[Abstract/Free Full Text]
  59. Milani, M., Pesce, A., Ouellet, Y., Dewilde, S., Friedman, J., Ascenzi, P., Guertin, M., and Bolognesi, M. (2004) J. Biol. Chem. 279, 21520-21525[Abstract/Free Full Text]
  60. Jentzen, W., Song, X. Z., and Shelnutt, J. A. (1997) J. Phys. Chem. B 101, 1684-1699
  61. Tilton, R. F., Jr., Kuntz, I. D., Jr., and Petsko, G. A. (1984) Biochemistry 23, 2849-2857[CrossRef][Medline] [Order article via Infotrieve]
  62. Teeter, M. (2004) Protein Sci. 13, 313-318[CrossRef][Medline] [Order article via Infotrieve]
  63. Milani, M., Pesce, A., Ouelett, Y., Ascenzi, P., Guertin, M., and Bolognesi, M. (2001) EMBO J. 20, 3902-3909[CrossRef][Medline] [Order article via Infotrieve]
  64. Pesce, A., Nardini, M., Dewilde, S., Geuens, E., Yamauchi, K., Ascenzi, P., Riggs, A. F., Moens, L., and Bolognesi, M. (2002) Structure 10, 725-735[Medline] [Order article via Infotrieve]
  65. Milani, M., Pesce, A., Bolognesi, M., Bocedi, A., and Ascenzi, P. (2003) Biochem. Mol. Biol. Educ. 31, 228-233
  66. Bolognesi, M., Cannillo, E., Ascenzi, P., Giacometti, G. M., Merli, A., and Brunori, M. (1982) J. Mol. Biol. 158, 305-315[CrossRef][Medline] [Order article via Infotrieve]
  67. Perutz, M. F. (1989) Trends Biochem. Sci. 14, 42-44[CrossRef][Medline] [Order article via Infotrieve]
  68. Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752[Abstract/Free Full Text]
  69. Giacometti, G. M., Ascenzi, P., Brunori, M., Rigatti, G., Giacometti, G., and Bolognesi, M. (1981) J. Mol. Biol. 151, 315-319[CrossRef][Medline] [Order article via Infotrieve]
  70. Coletta, M., Angeletti, M., de Sanctis, G., Cerroni, L., Giardina, B., Amiconi, G., and Ascenzi, P (1996) Eur. J. Biochem. 235, 49-53[Medline] [Order article via Infotrieve]
  71. Couture, M., Das, T. K., Lee, H. C., Peisach, J., Rousseau, D. L., Wittenberg, B. A., Wittenberg, J. B., and Guertin, M. (1999) J. Biol. Chem. 274, 6898-6910[Abstract/Free Full Text]
  72. Weiland, T. R., Kundu, S., Trent, J. T., III, Hoy, J. A., and Hargrove, M. S. (2004) Am. Chem. Soc. 126, 11930-11935
  73. Kamimura, S., Matsuoka, A., Imai, K., and Shikama, K. (2003) Eur. J. Biochem. 270, 1424-1433[Medline] [Order article via Infotrieve]
  74. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
  75. Bashford, D. (1999) in Scientific Computing in Object-oriented Parallel Environments (Ishikawa, Y., Oldehoeft, R. R., Reynders, J. V. W., and Tholburn, M., eds) Vol. 1343 of Lecture Notes in Computer Science, pp. 233-240, ISCOPE97, Springer, Berlin

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Exp. Biol.Home page
N. Heymann and F.-O. Lehmann
The significance of spiracle conductance and spatial arrangement for flight muscle function and aerodynamic performance in flying Drosophila
J. Exp. Biol., May 1, 2006; 209(9): 1662 - 1677.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/29/27222    most recent
M503814200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de Sanctis, D.
Right arrow Articles by Bolognesi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Sanctis, D.
Right arrow Articles by Bolognesi, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement