Vol. 187, No. 2, 1992 September 16, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1063-l 070 STRUCTURAL STUDY AND PRELIMINARY CRYSTALLOGRAPHIC DATA FOR THE HEMOGLOBIN FROM REINDEER (Rangifer tarandus tarandus) Elena Contia, Elena Casalea, Paolo Ascenzt ‘h, Massimo Colettae, Saverio G. Condo’d, Angelo Merlic, Bruno Giardinaf, Domenico Bordog, and Martin0 Bolognesi aZ a Dipartimento di Genetica e Microbiologia, Universita di Pavia, Via Abbiategrasso, 207, 27100 Pavia, Italy h Dipartimento di Scienza e Tecnologia de1 Farmaco, Universita di Torino, Via Pietro Giuria, 9, 10125 Torino, Italy C Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita di Camerino, Via Filippo Camerini, 2, 62032 Camerino, Italy d Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita di Roma “Tor Vergata”, Via Orazio Raimondo, 00173 Rome, Italy e Istituto di Scienze Biochimiche, Universith di Parma. Viale delle Scienze, 43 100 Parma, Italy f Istituto di Chimica, CNR Centro di Chimica dei Recettori, Universith Cattolica de1 Sacro Cuore. Largo Francesco Vito, 1, 00176 Rome, Italy g Gruppo Biostrutture, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genova, Italy Received July 31, 1992 SUMMARY. The ferric form of reindeer hemoglobin (Rangifer farand~ tarandus) has been crystallized in an orthorhombic crystalline form from polyethylene glycol solutions, at pH 8.2. The crystals belong to the orthorhombic space group P212 12 1, with unit cell edges a = 84.2 A, b = 59.9 A, c = 119.5 A; one hemoglobin tetramer is contained in the asymmetric unit. The crystals diffract X-rays to a limit spacing of 3.0 A. Inspection of amino acid sequences in the N-terminal region of P-chains, and analysis of hemoglobin three-dimensional models, allows one to Abbreviations used: Hemoglobin, Hb; 2,3-diphosphoglycerate, 2,3-DPG; polyethyleneglycol, PEG. The positions occupied by amino acid residues in hemoglobin molecules have been indicated by their chain type (i.e. a or p), by the sequence number, and according to the topological convention of Perutz (24).
Transcript
Vol. 187, No. 2, 1992
September 16, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1063-l 070
STRUCTURAL STUDY AND PRELIMINARY CRYSTALLOGRAPHIC
DATA FOR THE HEMOGLOBIN FROM REINDEER
(Rangifer tarandus tarandus)
Elena Contia, Elena Casalea, Paolo Ascenzt ‘h, Massimo Colettae, Saverio G. Condo’d, Angelo Merlic, Bruno Giardinaf, Domenico Bordog, and
Martin0 Bolognesi aZ
a Dipartimento di Genetica e Microbiologia, Universita di Pavia, Via Abbiategrasso, 207, 27100 Pavia, Italy
h Dipartimento di Scienza e Tecnologia de1 Farmaco, Universita di Torino, Via Pietro Giuria, 9, 10125 Torino, Italy
C Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita di Camerino, Via Filippo Camerini, 2, 62032 Camerino, Italy
d Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita di Roma “Tor Vergata”, Via Orazio Raimondo, 00173
Rome, Italy e Istituto di Scienze Biochimiche, Universith di Parma. Viale delle Scienze,
43 100 Parma, Italy f Istituto di Chimica, CNR Centro di Chimica dei Recettori, Universith
Cattolica de1 Sacro Cuore. Largo Francesco Vito, 1, 00176 Rome, Italy
g Gruppo Biostrutture, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genova, Italy
Received July 31, 1992
SUMMARY. The ferric form of reindeer hemoglobin (Rangifer farand~ tarandus) has been crystallized in an orthorhombic crystalline form from polyethylene glycol solutions, at pH 8.2. The crystals belong to the orthorhombic space group P212 12 1, with unit cell edges a = 84.2 A, b = 59.9 A, c = 119.5 A; one hemoglobin tetramer is contained in the asymmetric unit. The crystals diffract X-rays to a limit spacing of 3.0 A. Inspection of amino acid sequences in the N-terminal region of P-chains, and analysis of hemoglobin three-dimensional models, allows one to
Abbreviations used: Hemoglobin, Hb; 2,3-diphosphoglycerate, 2,3-DPG; polyethyleneglycol, PEG. The positions occupied by amino acid residues in hemoglobin molecules have been indicated by their chain type (i.e. a or p), by the sequence number, and according to the topological convention of Perutz (24).
Vol. 187, No. 2, 1992 BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rationalize, on a molecular basis, the reduced 02 affinity and the decreased effect of organic phosphates observed in ruminant hemoglobins. By analogy, the analysis is extended to birds and reptiles, whose hemoglobin P-chains display, as in ruminants, the deletion of the N-terminal residue and a methionine at the NA2 position. I 199~ ~~~~~~~~ pTfSS,
Ruminant hemoglobins are characterized by an 02 affinity which is
lower than that observed in Hb’s from most mammals, including human HbA (1). This functional difference is enhanced in the presence of chloride ions, whereas the effect of organic phosphates, like 2,3-DPG, on 02 affinity
is reduced with respect to human HbA (2,3). Moreover, the functional
interplay between 2,3-DPG and Cl-, in ruminant Hb’s, brings about a situation whereby the 02-linked effect of either one is cancelled in the
presence of saturating amounts of the other effector (3). Furthermore, a
very recent functional characterization of the Hb from reindeer (Rangifer
tarandus tarandus) has shown that, differently from several other mammalian Hb’s (for which the shape of the 02 binding curve appears to
be independent of temperature over a large range of oxygen saturation
levels (4)), temperature in this Hb acts mainly on the shape of the binding curve, the overall 02 affinity being scarcely affected (5,6). In particular, a
virtual temperature independence of the upper asymptote,
representative of the R state of the molecule, is observed. To our knowledge, this is the first time that the R state of a Hb is found to display such a small AH for ligand binding. Conversely, the lower
asymptote, representative of the T state of the molecule, is affected by temperature, being characterized by a strong exothermic character for 02
binding (6). From this thermodynamic analysis, the overall enthalpy change for 02 binding turns out to be very small, and the reaction appears
to be almost totally entropy-driven. This finding has been interpreted in
relation to the wide range of temperatures (from -40” C to +20” C) that reindeer encounters in the wilderness (7). In fact, by virtue of the very small AH of 02 binding, oxygen delivery is not drastically impaired at the
level of the peripheral tissues, which may be up to 10’ C cooler than the lungs. Such a low value for the oxygenation enthalpy, which reindeer
shares with other Hb’s from arctic ruminants (such as musk ox, see S), can also be observed in ruminants living at more temperate climates, like ox
(7). This common behaviour, which is possibly indicative of similar structural characteristics for this class of hemoproteins, at present can
only be partially interpreted on the basis of primary structures (7,9). Reindeer Hb, thus, may be thought to represent a type case of a large class of Hb’s characterized by (i) low 0 2 affinity, (ii) low exothermic
oxygenation enthalpy, and (iii) a reduced effect of organic phosphates. A
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structural characterization of this Hb is therefore justified, being in line
with our previous investigations on different globin structures (10,l l),
and may help in unraveling the molecular bases of the peculiar functional
behaviours observed in ruminant Hb’s.
MATERIALS AND METHODS
Crystal Data. Reindeer Hb was purified as previously reported (3,12). The purified protein was oxidized to the ferric form by dialysis against 0.005 M ferricyanide solutions in 0.05 M phosphate buffer, at pH 7.0, for 24 hours at 4°C. The physico-chemical conditions affecting crystal growth were explored using the vapour-diffusion technique (13). The crystallization tests were assembled equilibrating a 5-20 1.11 protein droplet, containing reindeer ferric Hb at 10 mg/ml in 0.05 M tris buffer, 9% (w/v) PEG (6,000 average Mr), against a 1 ml reservoir solution in the same buffer medium, containing 18% PEG. The best crystals were found to grow at pH 8.2, in about one week, at 4’ C. The crystals showed prismatic habitus, growing to a largest size of 0.2x0.2x1.0 mm3. After recovery of the crystals, and storage in a stabilizing solution at 25% PEG, they were analyzed by means of conventional X-ray precession photographic methods. From inspection of the systematic absences and of the symmetry of the recorded reciprocal lattice planes, the crystals were shown to belong to the orthorhombic space group P21212 1, with unit cell edges a = 84.2 A, b = 59.9 A, c = 119.5 A. Assuming a molecular mass of 64,000 Dalton for the tetrameric reindeer Hb molecule, the crystal packing parameter Vm is 2.35 A3/Da if a whole tetramer is contained in the asymmetric unit (14); the corresponding (volume) solvent content of the crystals is 48%. Crystals of the aquo-met Hb form could be converterd to the Hb:cyanide complex by soaking in stabilizing solutions containing saturating levels of the ligand. Moreover, under the same growth conditions, isomorphous crystals of the reindeer Hb:cyanide complex could be grown. Freshly grown crystals show measurable diffracted intensities up to 3.0 A resolution on conventional precession photographs.
Microspectrophotometry. Small single crystals of reindeer methemoglobin and met-Hb:CN adduct were spectroscopically characterized by polarized light single crystal microspectrophotometry, using a U.V.-visible Zeiss MPM-03 apparatus. Absorption spectra were recorded between 450 and 750 nm with the incident light beam polarized parallel to the direction of the optical axes. The spectrum of the crystalline met-Hb:CN adduct, recorded at pH 8.2, is reported in Figure la, and is identical to the spectrum in solution. Spectra of met-Hb single crystals were recorded at pH 8.2 and, after equilibration in stabilizing solutions, at pH 6.0 and 9.0. The spectra recorded at pH 8.2 and 9.0 are identical and typical of hydroxy-met-Hb. At pH 6.0 the spectrum is characteristic of aquo-met-Hb (Figure lb). Crystals were also equilibrated with a stabilizing solution containing sodium azide and the spectrum of the adduct metHb:Nj- was recorded.
Crystalline reindeer Hb samples were reduced by means of sodium dithionite and the spectrum of deoxy-Hb showed a maximum at 555 nm.
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Figure 1. Single crystal absorption spectra of reindeer ferric Hb:cyanide adduct (a), and of the aquo derivative of the protein (b) at pH 6.0 (dashed line) and at pH 8.2 (continuous line).
After equilibration with an oxygenated solution the crystal showed the two bands at 540 and 575 nm, typical of oxyhemoglobin. During these reactions the crystals did not break nor dissolve.
The spectra of all the crystalline derivatives examined show absorption maxima at the same wavelenghts as the corresponding derivatives in solution (see Table 1). All the spectra show very little polarization of the absorption bands, suggesting a special orientation of the hemes with respect to the crystallographic axes.
RESULTS AND DISCUSSION
Concerning the three-dimensional structural features that are expected to characterize reindeer Hb (and possibly ruminant Hb’s in general), a particular attention deserves the organic phosphate binding pocket, located between the N-termini of the two p-chains of the
functional tetramer. As mentioned above, reindeer and all other ruminant
Hb’s are only weakly modulated by 2,3-DPG, expecially considering the low level of this organic phosphate in ruminant erythrocytes (about 0.4 mM) and the comparable role, in quantitative terms, played by chloride ions, present in normal levels (about O.lM (3,5,6)).
Inspection of the aminoacid sequences, and model building of 2,3-
DPG binding pocket (15), provides a structural description of the molecule which supports functional observations. Indeed, as for all ruminant Hb sequences known so far (16), in reindeer Hb residue Val j3 I(NAI) is deleted, and His /32(NA2) is replaced by Met (4,17). Thus, two positive
charges from histidines, and involved in organic phosphate binding, are
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Table 1 . Extinction coefficients of the ferric and ferrous derivatives of reindeer Hb (T = 20°C)
Hb derivative E (mM-I cm-l) ?L max (nm) PH
Ferric Hb (aquo-met)a
I! II
10.1
4.5
Ferric Hb (hydroxy-met)b 11.2
11 81 9.3
Hb:azidea
11 11
12.6 540
9.8 574
Hb deoxya 12.8 555 7.0
Hb:Oza
11 11
13.8 540
14.4 575
499 6.0
630 6.0
540
575
9.0
9.0
7.0
7.0
7.0
7.0
a : 0.1 M phosphate buffer b : 2% borate buffer
removed, with consequent alteration of the electrostatics of effector
binding; moreover, the two N-termini are displaced, creating a potentially wider binding pocket. Figure 2 shows a hypothetical model of the 2,3-DPG
binding site for reindeer Hb, which has been modeled using the molecular graphics program FRODO (18), and as starting reference the atomic co- ordinates (19) of human deoxy HbA (20), and ruminant sickling deer type
III Hb, for which a partially refined crystal structure is available (21). According to Perutz and Imai (1) and Ascenzi et al. (17), Met @2(NA2) can
be expected to stabilize the low affinity quaternary structure of reindeer Hb (as well as of other ruminant Hb’s) via a hydrophobic interaction involving Leu P3(NA3), Leu P78(EF2), Leu p8l(EF5), and Val p133(Hll).
Such an interaction is not possible in “non-ruminant-like” sequences,
where residue NA2 is a polar His, even when NA3, EF2, EF5 and Hll
apolar residues are conserved (see Figure 2). The particular structure of
the N-terminal amino acid sequence, present in all ruminant Hb’s, and its interaction with specific hydrophobic regions of the molecule, seems to
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b
Figure 2. Stereo view of the 2,3-DPG binding site in human Hb (a), and in reindeer Hb (b), showing residues provided by the two p chains in the tetramer. The human Hb structure was obtained from data reported by Fermi et al. (20). The reindeer Hb model has been prepared on the basis of human deoxy Hb (20) with proper amino acid substitutions (23).
account for both the intrinsically reduced 02 affinity and the weak effect
of organic phosphates on the functional properties of this class of hemoglobins. Thus both the deoxy and the liganded conformational states of reindeer Hb are characterized by a lower affinity constant for 2,3-DPG
with respect to that observed for the corresponding conformations of
human HbA (6).
A systematic search on amino acid sequences shows that residue Met @2(NA2) is also present in birds, reptiles, platypus and in tuatara Hb l3 chains; moreover, all these non-ruminant amino acid sequences show
the deletion of the N-terminal Val pl(NA1) residue (16). Inspection of
residues expected to face the NA2 site in the three-dimensional structures shows that in these Hb’s residues EF2, EF5, and Hll are Leu, Leu and Phe, respectively. Thus, the hydrophobic character of the site, whose geomentry is only in part altered by the Val l3133(Hll) --> Phe
substitution, is kept. In this context, it is worthwhile noticing that reptile and platypus Hb’s display low 02 intrinsic affinity, which, in turn, is almost unaffected by the presence of organic phosphates (22). Moreover, the spectroscopic properties of the nitric oxide derivative of reptile Hb’s are in keeping with the low affinity conformation of the hemoprotein (22). A peculiar example in this series is the adult loggerhead sea turle Hb, whose N-terminal Met p2(NA2) residue is acetylated (22), thus
further depleting the 2,3-DPG binding pocket of two positive charges.
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ACKNOWLEDGMENT
We wish to thank Dr. Giulio Fermi, from the MRC Laboratory of Molecular Biology, Cambridge (UK), for making available the atomic coordinates of the 2,3 DPG binding pocket in human Hb, which resulted from his crystallographic and modeling studies.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
REFERENCES
Peru& M.F. and Imai, K. (1980) J. Mol. Biol. 136, 183-191.
Fronticelli, C., Bucci, E. and Razynska, A. (1988) J. Mol. Biol. 202, 343-348.
Giardina, B., Brix, O., Colosimo, A., Petruzzelli, R., Cerroni, L. and Condo’, S.G. (1990) Eur.J.Biochem. 194, 61-65.
Imai, K. and Yonetani, T. (1975) J. Biol. Chem. 250, 7093-7098.
Giardina, B., Brix, O., Nuutinen, M., El Sherbini, S., Bardgard, A., Lazzarino, G. and Condo, S.G. (1989) FEBS Lett. 247, 135-138.
Giardina, B., Condo, S.G., Petruzzelli, R., Bardgard, A. and Brix, 0. (1990) Biophys. Chem. 37, 281-286.
Coletta, M., Clementi, M.E., Ascenzi, P., Petruzzelli, R., Condo, S.G. and Giardina, B. (1992) Eur. J. Biochem. 204, 115.5-1157.
Brix, O., Bardgard, A., Mathisen, S., El Sherbini, S., Condo, S.G. and Giardina, B. (1989) Comp. Biochem. Physiol. M, 135-138.
Fronticelli, C. (1990) Biophys. Chem. 37, 141-146.
Bolognesi, M., Onesti, S., Gatti, G., Coda, A., Ascenzi, P. and Brunori, M. (1989) J. Mol. Biol. 205, 529-544.
Casale, E., Lionetti, C., Coda, A., Merli, A., Ascenzi, P., Wittenberg, J.B. and Bolognesi, M. (1991) J. Mol. Biol. 222, 447-449.
Antonini, E. and Brunori, M. (1971) in “Hemoglobin and Myoglobin in their Reactions with Ligands”. A. Neuberger and E.L. Tatum, Eds., North-Holland Publishing Co., Amsterdam.
McPherson, A. (1982) in “Preparation and Analysis of Protein Crystals”, John Wiley and Sons, New York.
Matthews, B.W. (1974) J. Mol. Biol. 82, 513-526.
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(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
Arnone, A. (1972) Nature 237, 146-149.
Kleinschmidt, T. and Sgouros, J.G. (1987) Biol. Chem. Hoppe-Seyler 368, 579-615.
Ascenzi, P., Coletta, M., Desideri, A., Petruzzelli, R., Polizio, R., Bolognesi, M., Condo’, S.G. and Giardina, B. (1992) J.Inorg.Biochem, 45, 31-37.
Jones, T.A. (1978) J. Appl. Crystallogr. 11, 268-272.
Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F.Jr., Brice, M.D., Rodgers, J.R., Kennard, O., Shimanouchi, T. and Tasumi, M. (1977) J. Mol. Biol. 112, 535-542.
Fermi, G., Perutz, M.F., Shaaman, B. and Fourme, R. (1984) J. Mol. Biol. 175, 159-174.
