From the Department of Chemistry and Biochemistry,
University of Texas at Austin, Austin, Texas 78712 and the
§ Department of Zoology, University of Texas at Austin,
Austin, Texas 78712
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ABSTRACT |
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Oxygen binding by chicken blood shows enhanced
cooperativity at high levels of oxygen saturation. This implies that
deoxy hemoglobin tetramers self-associate. The crystal structure of an
R-state form of chicken hemoglobin D has been solved to 2.3-Å resolution using molecular replacement phases derived from human oxyhemoglobin. The model consists of an
The oxygen binding curves of chicken blood samples taken at
different stages of development exhibit Hill coefficients which exceed
4.0 (super-cooperativity) at oxygen saturation levels between 70 and
90% oxygenation (1-5). Elevated levels of oxygen-binding cooperativity should be advantageous because such hemoglobins (Hbs)1 would deliver a
greater quantity of oxygen to the tissues under normal physiological
conditions. Super-cooperativity has also been observed in the blood and
Hbs of several species of amphibians (6-8), reptiles (9-10), other
birds (1, 11), and embryonic mammals (12-14). Although incomplete
oxygen saturation resulted in spuriously high Hill coefficients in much
of the early data (see Refs. 15-18), the data of Lapennas and Reeves
(5) on chicken blood and Holland et al. (12-14) on
embryonic marsupial blood were obtained at saturating pressures of
O2.
The blood of adult chickens, as in most other birds, is composed
of two Hb components that have identical The cooperative binding of oxygen by tetrameric Hbs arises from
the transition of the Hb from a conformation with a low oxygen affinity
(T-state) to a conformation with a high oxygen affinity (R-state) (21,
22). Much of our understanding of this process has come from the
crystallographic analysis of the unligated (T-state) and ligated
(R-state) structures of both human Hb A (23-26) and horse Hb (27-30).
Although a structural explanation of super-cooperativity requires the
crystallographic analysis of both the R- and T-state forms of chicken
Hb D, the analysis of intermediate structures are likely to be
necessary for understanding the mechanism of cooperativity because
knowledge of two thermodynamic states does not itself determine the
pathway between them. We report here the structure of an R-state form
of chicken Hb D solved with diffraction data to 2.3-Å resolution. This
structure provides the first step towards a model for enhanced
cooperativity and is the first structure reported for the minor Hb (D)
component of birds, and the second structure of an avian Hb (31). The
re-analysis of the oxygen binding data in the literature demonstrates a
previously, overlooked relationship between super-cooperativity and
inositol hexaphosphate (IHP) binding.
Purification, Crystallization, and Data
Collection--
Chicken Hb D was purified from chicken blood using
DEAE and gel filtration chromatography as described previously (20). Several grains of sodium dithionite (Miles-Platting, Manchester, United
Kingdom) were added to the Hb solutions prior to crystallization. Conditions for crystallization were screened by the sparse matrix technique (32) with the hanging drop method (33). Chicken Hb D was
crystallized with a combination of PEG 3350 and either lithium sulfate
or sodium acetate as the precipitant. The precipitant concentration was
varied from 20 to 30% polyethylene glycol and from 10 to 30 mM salt, with a protein concentration of 20 mg/ml in 50 mM Tris/HCl, pH 7.5. The crystal setups were prepared under nitrogen in a glove box (Bactron 1 model, Anaerobe Systems Inc.) and
then transferred to a desiccator that was flushed with nitrogen. The
desiccator was then placed in a cold room at 4 °C for 2 weeks. Crystals of Hb D grew as plates of about 0.1 mm in thickness and 1.0-mm wide.
X-ray data were collected on a San Diego Multiwire area detector
equipped with a Rigaku RU-200 rotating anode generator operated at 50 kV and 110 mA. The initial x-ray analysis indicated that the chicken Hb
D crystals belong to the space group P21 with cell dimensions of a = 53.96 Å, b = 80.51 Å, c = 82.11 Å, and Structure Determination--
The initial phases were obtained by
the molecular replacement method with the human Hb tetramer serving as
the search model. The Refinement--
After the initial molecular replacement
solution, a set of 3407 reflections or 10% of all the reflections was
set aside to be used in the calculation of R-free (38). The
model was then subjected to an additional round of rigid body
refinement in which the entire model was first refined as a rigid body
before the individual subunits were refined as rigid bodies. The
chicken Hb D model was then subjected to 8 additional rounds of X-PLOR refinement using the ideal parameter set of Engh and Huber (39). During
the first five rounds of X-PLOR refinement, the two
A typical round of X-PLOR refinement included positional
refinement using the conjugate gradient energy minimization (40) followed by B-factor refinement. Early in the refinement
process (rounds 1-3), both a main chain and a side chain
B-factor were optimized for each residue. After the third
round, individual B-factors were refined. The first round of
refinement included the refinement of each of the helical segments as
rigid bodies. The second and third rounds included a simulated
annealing step (41, 42). Between each round of X-PLOR refinement, the
model was fitted to either 2Fo
The first round of X-PLOR refinement resulted in a model with an
R-factor of 0.260 (R-free = 0.309) for all
measured reflections between 8.0- and 3.0-Å resolution. The quality of
the electron density map allowed the side chains of 80 residues (about
one-half of the residues truncated) to be added to the model. This
model was then subjected to a second round of refinement that extended the data to 2.3-Å resolution over 10 steps of energy minimization. Following the simulated annealing and group B-factor
refinement steps, the R-factor rose to 0.261 and the
R-free dropped to 0.296 for all measured reflections between
8.0- and 2.3-Å resolution. After the fourth round of refinement, six
residues were removed from and 52 water molecules were added to the
model. Water molecules were added into Fo Analysis of the Model--
Least-squares superposition
calculations were performed using LSQMAN (47) and the results were
displayed in O. The coordinates for bar-headed goose Hb A
was kindly provided by Dr. Jeremy Tame (University of York, United
Kingdom). The plane of the heme group was calculated using the program
PLANES (46). Subunit contacts were measured using CONTACT (46). The
surface area was determined using the method of Lee and Richards (48) as implemented in SURFACE (46). Figs. 1, 2, and 4 were prepared with
MOLSCRIPT (49). Fig. 3 was prepared with the program MOLVIEW (50). Fig.
10 was prepared with GRASP (51). The sequence analysis included the
primary structures of 20 The Refined Model--
Crystals of chicken Hb D belong to the
monoclinic space group P21 with one
Noncrystallographic Symmetry--
The presence of an
Overall Structure and the Comparison with Human Oxy-Hb
A--
Chicken Hb D has a quaternary structure which is consistent
with other R-state Hbs, but differs from other T-state or R2 state structures. The superposition of 570 C
As expected, the folds of the
Several other regions of the Comparison with Hb A of Bar-headed Goose--
The structure of one
other avian Hb (Bar-headed goose Hb A) has been reported, also in the
R-state (31). Although the The The The Heme Regions--
The heme irons in chicken Hb D appear to be
coordinated to either water or oxygen. Initially, it was assumed that
the R-state model represented the aquomet form of Hb D and water
molecules were modeled into the electron density located near the heme
iron from each Hb subunit. Following four rounds of refinement and model fitting, each water moved to a position that is 2.9 Å away from
the iron, allowing the water to hydrogen bond to the distal histidine.
Although this distance is consistent with that observed in the
structure of the aquomet form of Chironomous thummi Hb (55),
the water-to-iron distance of chicken Hb D differs from the
corresponding values observed both in the structure of horse aquomet Hb
(30) and in the high resolution structures of ferric porphyrins
complexed with water ligands (56, 57). Re-examination of the electron
density from a set of simulated annealing 2Fo
The heme groups of the Inositol Hexaphosphate-binding Site--
Organic phosphates lower
the oxygen affinity of vertebrate Hbs by preferentially binding to the
T-state of the tetramer (58, 59). The major organic phosphate in human
red blood cells is bisphosphoglycerate, whereas that in chicken
erythrocytes is inositol pentaphosphate (60). Most in vitro
studies utilize the more readily available IHP. Both human and chicken
Hbs bind IHP in a maximum ratio of 1 molecule of IHP per Hb tetramer
(61). The crystallographic analysis of human Hb complexed with IHP
indicates that IHP binds in a surface cavity formed between the two
Chicken Hbs A and D bind organic phosphates more tightly than do
mammalian Hbs. The oxy and deoxy forms of human Hb at pH 6.8 bind
bisphosphoglycerate with association constants of 1.2 × 103 M
The organic phosphate-binding site of the R-state of chicken Hb D
does not allow IHP to fit easily into its normal binding site. A
reasonable model of chicken oxy-Hb with IHP can be made by moving the
organic phosphate 4.5 Å toward the Hb surface, enabling IHP to
interact with the amino terminus as well as the side chains of
Lys Relationship between IHP and Super-cooperativity--
Although a
number of oxygen binding studies have been performed on purified
chicken Hbs, there are no reports of super-cooperativity in
vitro. The lack of in vitro evidence for
super-cooperativity of adult Hbs prompted us to re-examine the
published oxygen binding curves of chicken Hbs A and D (54). The
original oxygen binding data for chicken Hbs A and D were kindly
provided by Dr. R. E. Isaacks. This particular data set was
collected using the continuous recording method of Longmuir and Chow
(65) with the modifications described by Lian et al. (66).
Hbs A and D were purified by anion exchange chromatography. The
oxygen-binding curves for Hbs A and D were measured in both the
presence and absence of 2 mM IHP (54) at a Hb concentration
of 1.7 mg/ml.
The original analysis of Isaacks et al. (54) reported Hill
coefficients at 50% saturation. However, the functionally relevant values in Hbs which self-associate occur at higher oxygenation levels
(67). Therefore, the O2-binding curves of chicken Hbs D and
A were re-evaluated. The Hill coefficients were calculated as a
function of oxygen saturation. Separate least-squares lines were fitted
to the low oxygen saturation (<55% saturation) and high oxygen
saturation (
It should be mentioned that the oxygen-binding measurements of Isaacks
et al. (54) suffer from two possible sources of error. The
first possible source of error is due to the assumption that both the
production of oxygen by the electrode and the consumption of oxygen by
beef heart mitochondria are linear processes. The original analysis of
Longmuir and Chow (65) demonstrated that both of these assumptions were
true in the absence of organic phosphates. However, it is not known if
both the oxygen production and oxygen consumption processes of this
experiment are truly linear when IHP is added to the sample. The second
source of possible error is due to an incomplete saturation of the Hb
sample. It has been demonstrated that artificially high Hill
coefficients occur when the 100% saturation point is underestimated
(15-17). This problem is particularly applicable to chicken Hbs
because of their low oxygen affinity. The 100% saturation point for
the oxygen binding analysis of Isaacks et al. (54) was
estimated with oxygen saturation points measured at relatively high
oxygen pressures (~265 torr). The 100% saturation point of this
analysis can be underestimated by values between 4 and 7% without
reducing the Hill coefficients of the Hb D-IHP and Hb A-IHP samples to values below 4.0.
The Interface between Deoxy-Hb D Tetramers--
The
interface between two Hb D tetramers originally was thought to involve
the EF corners from the
The interface between two deoxy-Hb D tetramers should resemble
that observed in the structures of other oligomeric proteins. Analysis
of the high-resolution structures of 23 oligomeric proteins shows that
a higher percentage of hydrophobic residues are found at the subunit
interfaces than are normally located on the surface of the protein (68,
69). Therefore, large, hydrophobic residues (Leu, Ile, Met, Val, Cys,
Tyr, and Trp) which are located on the surface of Hb D might contribute
to the formation of the tetramer-tetramer interface. Table
III lists the 10 hydrophobic residues
that have at least 40 Å2 of solvent-exposed surface area.
The alignments of the
An alternative method for locating the tetramer-tetramer interface is
to analyze the electrostatic surface potential for chicken deoxy-Hb D. A hypothetical deoxy model was constructed by superimposing each of the
The surface formed by parts of the D and E helices of the Possible Mechanism for
Super-cooperativity--
Super-cooperativity in chicken Hbs depends on
the formation of a tetramer-tetramer interface which further shifts the
conformation to a lower oxygen-affinity state and thus could mediate
cooperativity additional to that observed within the
2
2 tetramer in the asymmetric unit
and has been refined to a R-factor of 0.222 (R-free = 0.257) for 29,702 reflections between 10.0- and 2.3-Å resolution. Chicken Hb D differs most from human
oxyhemoglobin in the AB and GH corners of the
subunits and the EF
corner of the
subunits. Reanalysis of published oxygen binding data
for chicken Hbs shows that both chicken Hb A and Hb D possess enhanced
cooperativity in vitro when inositol hexaphosphate is
present. The electrostatic surface potential for a calculated model of
chicken deoxy-Hb D tetramers shows a pronounced hydrophobic patch that
involves parts of the D and E helices of the
subunits. This
hydrophobic patch is a promising candidate for a tetramer-tetramer
interface that could regulate oxygen binding via the distal histidine.
INTRODUCTION
Top
Abstract
Introduction
References
chains but differ in the
sequences of the
subunits. Hb A
(
A2
2) and Hb D
(
D2
2) are expressed in a 3:1
ratio in adult chickens (19). The sequence of the
chain from Hb D
(
D) is 58.9% identical to that of Hb A
(
A). The large Hill coefficients observed in chicken
blood imply that the deoxy-Hb tetramers associate into oligomers
because the Hill coefficient cannot be greater than the number of
subunits. Although super-cooperativity has not been reported for the
purified adult chicken Hb components, the self-association of deoxy
tetramers is known. Sedimentation analysis of chicken Hbs A and D
showed that only deoxygenated Hb D self-associates. This analysis
demonstrated that deoxy-Hb D forms a dimer of tetramers with an
association constant of 1.26 × 104
M
1 (20). Because the two chicken Hb
components only differ in their
chains, it was concluded that the
tetramer-tetramer contacts lie on the surface of the
D
subunit (20). This led to a proposed model that describes the interface
that might form between two Hb D tetramers (20).
MATERIALS AND METHODS
= 104.51°. Assuming the
presence of two tetramers of 66,452 daltons in the unit cell, the
Matthew's parameter is equal to 2.6 Å3/dalton.
A data set was collected from nine orientations of a single crystal by
the method described by Xuong et al. (34). The data were
collected at room temperature, with a rotation angle of 0.12° in
,
and an exposure time of 60 s. The measured intensities were
processed with the University of California, San Diego package (35).
The resulting data set is 91% complete to 2.1-Å resolution with an
overall multiplicity of 6.8, and an overall Rsym
of 8.1%. Because of the low signal to noise in the last resolution
shell, only the data to 2.3-Å resolution were used in the refinement of the Hb D model. The last resolution shell (2.5-2.3 Å) has an average multiplicity of 3.6, a Rsym of 23.9%,
and an average I/
(I) of 2.3.
and
chains of chicken Hb D are 61.7 and
68.7% identical to those of human Hb A, respectively. Both the deoxy
(26) and the oxy (25) forms of human Hb were used as search models
because there was some doubt that the crystals of Hb D were deoxy. Each of the Hb models consisted of the human sequence with all identical residues being retained and all non-identical residues being truncated to alanine. All the molecular replacement and refinement routines were
performed using the computer program X-PLOR version 3.1 (36) running on
a DEC ALPHA server. The molecular replacement protocol included a
Patterson correlation refinement step (37) that allowed each of the
subunits of the search model to be optimized by rigid body refinement
before the translation function was performed. The cross-rotation
function followed by the Patterson correlation refinement using either
Hb search model resulted in a rotation solution at 288°, 45°,
348° (
1,
2,
3, zxz
Eulerian angles). The translation function for either model produced a
clear peak at 0.407, 0.000, and 0.232 (xyz in fractional coordinates).
An examination of the correctly positioned models indicated that the
conformation of both models described an R-state Hb; therefore, only
the oxy model, which gave a slightly better signal to noise ratio in
the translation function (12.4
/mean compared with 10.1
/mean for
the deoxy model) was refined against the observed data. The initial
R-factor of the positioned model was 0.457 for all measured
data between 8.0 and 3.0-Å resolution.
dimers were
constrained to be identical with strict non-crystallographic symmetry
(NCS). During the last three rounds of refinement, the regions of the
structure which showed asymmetry in the electron density between the
NCS-related subunits were allowed to refine without NCS restraints
whereas all other regions of the model were refined imposing tight NCS
restraints. During the last round of refinement, most of the main chain
atoms of the two dimers were tightly restrained whereas the side chain
atoms were loosely restrained. The first four and the last four
residues from each subunit as well as the residues that form the EF
corner from each subunit were not restrained. All attempts to
completely remove the NCS restraints resulted in a poorer model as
judged by an increase in R-free.
Fc
calc or Fo
Fc
calc electron density maps using
the program O (43) running on a Silicon Graphics Crimson
workstation. After each round of X-PLOR refinement and manual
intervention, the quality of the stereochemistry of the model was
monitored using the program PROCHECK (44). Regions of the structure
with either questionable density or poor geometry were rebuilt into
simulated annealing 2Fo
Fc and
Fo
Fc omit maps (45). The side
chains for the truncated residues were added to the model only if
suitable density was present in the 2Fo
Fc electron density maps.
Fc maps contoured at 3
using PEAKMAX and WATPEAK
(46). Water molecules were removed from the model if either the
2Fo
Fc density was poor or the
water failed to form at least one hydrogen bond with either a protein
atom or another water molecule. Following a round of energy
minimization and individual B-factor refinement, the
resulting model had a R-factor of 0.235 (R-free = 0.270). Additional waters were added to the
model during the last three rounds of refinement. The improvement of
the electron density during the last round of refinement allowed two of
the omitted residues to be added to the final model. Oxygen ligands
were modeled into simulated annealing omit maps before the last round
of X-plor refinement. The oxygen ligands were refined with tight
geometric restraints, but without NCS restraints.
A,
D, and
chains which were obtained from GenBankTM (52).
RESULTS
2
2 tetramer (a total of 574 residues) in
the asymmetric unit. The
D and
subunits of chicken
Hb D are composed of 141 and 146 amino acid residues, respectively. The
refined model of chicken Hb D consists of 570 amino acid residues, 4 heme groups, 141 water molecules, and three oxygen ligands. The last
residue of the
2 subunit (Arg141) and the
last three residues from the
1 subunit,
Lys144(HC1), Tyr145(HC2), and
His146(HC3) were omitted from the final model due to the
lack of electron density. In other areas of poor electron density, the
side chains of 4 residues were truncated to glycine, 25 were truncated
to alanine, and 6 were truncated by two or more atoms. The
R-factor of the refined model is 0.222 (R-free = 0.257) for all measured reflections between
10-2.3 Å resolution (Table I). The
geometry of the chicken Hb D model is excellent when compared with the ideal parameters of Engh and Huber (39). The Ramachandran plot indicates that 89.2% of the non-glycine, non-proline dihedral angles
lie in the most favored regions whereas 9.8 and 1% of the residues
have
and
angles which lie in the additionally allowed and
generous regions, respectively. No residue has dihedral angles in the
disallowed regions of the Ramachandran plot. The conformation of each
residue with
-
angles in the generous region of the Ramachandran
plot was verified by fitting each of these residues into simulated
annealing omit maps.
The refinement statistics of the current model of chicken oxy-Hb D
2
2 tetramer in the crystallographic
asymmetric unit results in the generation of a molecular 2-fold axis of
NCS between the two
dimers. Differences between the two dimers
are localized to small regions of the model which were not restrained
by NCS. The first and last 4 residues from both subunits show the
largest differences as indicated by average r.m.s. differences of 1.1 Å/C
atom. The residues which form the EF corner of the
subunits
(residues 75-77) show small differences as indicated by average r.m.s.
differences of 0.35 Å/C
atoms. The remaining C
atoms have
average r.m.s. differences of 0.03 Å/C
atom due to the tight
restraints. The side chain atoms show larger differences. Excluding the
first four and last 4 residues from each subunit, the superposition of
the
1
1 dimer onto the
2
2 dimer gives a r.m.s. difference of
0.47 Å for 2039 main chain and side chain atoms.
atoms from chicken Hb D onto
the corresponding C
atoms of human oxy-Hb (25) gives an r.m.s. value
of 1.0 Å. The superposition of the C
atoms from chicken Hb D onto
the corresponding atoms from either T-state or R2-state human Hb shows
larger differences. The r.m.s. values are 2.4 and 1.7 Å for the
superposition of 570 C
atoms from chicken Hb D onto the C
atoms
from human T-state (26) and R2-state (53) Hbs, respectively.
and
subunits of Hb D are
both similar to those of human Hb, with each
subunit composed of
seven helical segments (labeled A, B, C, E, F, G, and H) and each
subunit composed of eight helical segments (labeled A-H). The
inter-helical segments ("corners") formed between helical segments
are labeled according to the nomenclature used for myoglobin. The
superposition of 141 C
atoms of
1, 141 C
atoms of
2, 143 C
atoms of
1, and 146 C
atoms of
2 of human oxy-Hb (25) onto the corresponding
subunits of Hb D gives r.m.s. differences of 1.1, 0.85, 0.79, and 0.78 Å, respectively (Figs. 1 and
2). Both subunits of Hb D show moderate
differences in the positioning of the four NH2-terminal and
four COOH-terminal residues. However, these segments in chicken Hb D
also show the largest differences between NCS related subunits,
reflecting the increased flexibility of the termini from each R-state
Hb subunit. If these residues are excluded, 133 C
atoms of the
subunit and 138 C
atoms of the
subunit from human Hb superimpose
upon their counterparts in chicken Hb D with r.m.s. differences of 0.83 and 0.69 Å, respectively.
View larger version (42K):
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Fig. 1.
The stereo view of the subunit of human oxy-Hb (black) superimposed
upon the coordinates of chicken Hb D (gray). The
seven
helices are labeled A-C and E-H. The
superposition calculation included 140 C
atoms of the
2 subunit
and resulted in an r.m.s. difference of 0.85 Å. Note the differences
in the AB and GH corners between the two
subunits.
View larger version (40K):
[in a new window]
Fig. 2.
The stereo view of the subunit of human oxy-Hb (black) superimposed
upon the C
coordinates of chicken oxy-Hb D
(gray). The eight
helices are labeled
A-H. The superposition calculation included 146 C
atoms
of the
2 subunit and resulted in an r.m.s. difference of 0.78 Å.
Note the differences in the EF corners between the two
subunits.
and
subunits from chicken oxy-Hb D
also differ from the corresponding regions in human oxy-Hb. The AB
corner of the
subunits of Hb D forms a more helical turn than its
counterpart in human Hb (Fig. 1). This difference is due in part to the
substitution of 2 glycine residues in human Hb with
Ala
18(A16) and Glu
22(B3) in chicken Hb D. The tightness of this turn results in a slight change in the
orientation of helix B in the
subunit of chicken Hb D. The
replacement of a Pro
114(GH2) in human Hb with
Gly
114 in chicken Hb D results in a movement of the
bulge located in the middle of the GH corner. In chicken Hb D,
Gly
114 is oriented with a
angle of 42° and a
angle of
131°, causing a change in the orientation of this bulge,
shifting the GH corner, moving the C
atom of
Lys
115(GH3) by 3.7 Å, and positioning its side chain so
that it is rotated by 90° relative to that in human oxy-Hb. A third
difference between the two Hbs involves the rotation of the
COOH-terminal end of the E helices and of the EF corners (residues
73-84) of the
subunits so that the EF corner of the
subunit
from Hb D is closer to its A helix (Fig. 2).
chains of the two avian adult Hbs are
very similar (95.2% sequence identity), chicken
D is
only 56.0% identical to bar-headed goose
A, less than
the 61.7% identity which exists between human
and chicken
D. The tertiary structures of the
and
chains
from the two avian Hbs reflect the relative degree of sequence
identity. The superposition of 143 C
-subunit atoms from chicken
Hb D onto the corresponding atoms of the bar-headed goose Hb gives an
r.m.s. difference of 0.30 Å, compared with an r.m.s. difference of 0.7 Å for the superposition of 140 C
atoms in the
subunits. The
subunits of both bar-headed goose and chicken Hbs have nearly identical
AB corners but differ in the GH corners. The analysis of other avian
chain sequences suggests that the residues that comprise the AB
corner in both bar-headed goose and chicken Hbs are not absolutely
conserved in other avian Hbs. Although an acidic residue is present at
position 22 (B3) in 38 of the 40 avian
chains surveyed, only 15 of
these avian Hbs retain a glycine at position 18 (A16). Avian Hbs with
subunits that retain a glycine only at position 18 would be expected to have an AB corner that differs from that of chicken Hb D. The
D subunits of all but one avian Hb D include a
glycine at position 114 (GH2) whereas all 20 of the avian
A chains surveyed have GH corners with a proline at this
position. This result suggests that the conformation of the GH corner
observed in chicken Hb D will be characteristic of most Hb D components of avian blood.
1
1 Interfaces--
The
1
1 interface is similar to that found in
other vertebrate Hbs. Out of the 36 residues that form the
1
1 interface in chicken Hb D (based on
those residues with an atom which is within 4.0 Å of an atom from a
neighboring subunit), 21 (58%) are identical to their counterparts in
human Hb compared with 29 identities (81%) in bar-headed goose Hb A. Although most of these substitutions should not alter the stability of
the
1
1 interface, there are three
substitutions, Gly
114(GH2), Gln
103(G10),
and Ser
119(GH2), found in chicken Hb D which deserve
comment. The replacement of a proline by a glycine at
114 in chicken
Hb D results in the reorientation of the
114 main chain torsion
angle, thereby eliminating an intersubunit hydrogen bond. In place of
this H-bond, the
1
1 interface is
stabilized by two additional hydrogen bonds, one formed between the
hydroxyl group of Ser
119(GH2) and the carbonyl oxygen of
Val
111(G18) and a second between the side chains of
Gln
103(G10) and Asp
108(G10). The addition
of an extra H-bond should stabilize both the R- and T-states of chicken
Hb D. Gln
103 is conserved in all avian
D
sequences and about half of the avian
A sequences
whereas Asp
108 is conserved in all 20
sequences
surveyed. Ser
119 is only present in four of the avian
sequences.
a
2 Interface--
In human and horse
Hbs, the
1
2 interface involves a total of
17 residues, all of which are highly conserved in vertebrate Hbs.
During the T- to R-state transition, this interface acts as a switch.
Consequently, both the R- and T-state conformations have distinct sets
of interactions which contribute to the stabilization of the
1
2 interface. Like most R-state Hbs, the
1
2 interface of chicken Hb D includes a
hydrogen bond formed between Asp
94(G1) and
Asn
102(G4) and lacks a hydrogen bond between
Asn
97(G4) and Asp
99(G1). Like the
bar-headed goose Hb A, the
1
2 interface
of chicken Hb D substitutes Gln
38(C3) in place of a
threonine found in mammalian Hbs. The hydroxyl group of
Thr
38 in horse and human Hbs forms a hydrogen bond with
the main chain carbonyl oxygen of His
97(FG4) when the Hb
has an R-state conformation. The side chain of Gln
38 in
chicken Hb D cannot form an analogous hydrogen bond, thereby reducing
the stability of the
1
2 interface. This
substitution should increase the oxygen affinity of R-state chicken Hb
D because the loss of a hydrogen bond would result in an increase in
the concentration of
dimers which have high oxygen affinity. It is likely that Gln
38 would decrease the cooperativity of
chicken Hb D. Samples of purified chicken Hb D exhibit Hill
coefficients that are less than two in the absence of all organic
phosphates (54). Gln
38 is present in 27 out of the 40 avian
sequences surveyed.
Fc omit maps suggests that three of the four
subunits are ligated to oxygen and the fourth (the
2
subunit) is ligated to water (Fig. 3). An
additional round of refinement allowed the O-1 atoms of the oxygen
ligands of the
subunits to move to 1.8 Å from the heme iron and
the oxygen ligand of the
1 subunit to refine to 1.7 Å from the heme
iron. The orientation of the oxygen ligands from the two
subunits
differ. The Fe·O1·O2 angles for the
1 and
2 subunits are
138° and 173°, respectively. The water ligand of the
2 subunit refined to 1.8 Å from the heme iron, shorter
than the ~2.0 Å values observed in horse aquomet Hb (30). It would
be unusual for the two
subunits to have different ligands. The
ligand assignments based on the electron density at 2.3-Å resolution
must be considered as tentative. Nevertheless, this model provides the
best fit to the simulated annealing omit map (Fig. 3).
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Fig. 3.
The heme pockets for the
1 (top) and
2 (bottom) subunits. The
hemoglobin model is superimposed upon the electron density as
calculated from a
A weighted (75), simulated annealing
omit 2Fo
Fc map in which both
of the
heme groups were omitted from the map calculation. The
1
heme appears to be ligated to an oxygen molecule and the
2 heme is
best fit with a water as the ligand.
and
subunits of chicken Hb D resemble
those of the ligated forms of other vertebrate Hbs (Table II) in that the iron atoms all lie near
the plane of the heme atoms (defined by all heme atoms excluding those
in non-planar side chains). The iron atoms of each subunit are
approximately 0.2 Å out of the heme plane toward the proximal
histidine. These values can be contrasted to the corresponding
distances observed in human deoxy-Hb. In the absence of a ligand, the
hemes of deoxy-Hbs should have iron-to-heme plane distances between 0.5 and 0.6 Å. The heme groups of chicken Hb D, as in all known Hbs, are
ligated to the N
-2 of the proximal histidine from each subunit
(His87 in the
subunits and His92 in the
subunits). Each heme fits into a pocket lined with several hydrophobic
residues. In chicken Hb D, the heme pockets of the
subunits are
formed from 17 residues and those of the
subunits are formed from
16 residues. All of the residues that contact the heme groups in the
subunits are conserved in human Hb. The only difference between the
heme pockets of chicken and human Hbs is the substitution of
Ser
70(E14) in chickens with Ala
70 in
humans. This difference is probably not significant because serine is
found at this position in the
subunits of other mammalian Hbs. Thus
the heme pockets of chicken Hb D show no differences from other
vertebrate Hbs which could account for the super-cooperativity of
chicken blood.
The geometry of the heme groups from chicken oxy-Hb D, goose oxy-Hb A
(31), and human oxy-Hb (25)
dimers present in the asymmetric unit.
DISCUSSION
subunits, the same site which also binds bisphosphoglycerate (62). In this crystal structure, the IHP-binding site includes the amino terminus of Val
1(NA1) as well as the side chains of
His
2(NA2), Lys
82(EF7),
Asn
139(H17), and His
143(H21) from both
subunits (62).
1 and 1.7 × 104 M
1, respectively (63). The CO
R-state of chicken Hb A at pH 7.0 binds bisphosphoglycerate with an
association constant of 1 × 104
M
1 (61), which is approximately the same as
human deoxy-Hb. Similarly, the oxy form of a related avian Hb (Hb A
from bar-headed goose) binds IHP with an affinity similar to that of
human deoxy-Hb (64). Both the R- and T-states of chicken Hb bind IHP
more tightly than the equivalent forms of human Hb, although the IHP
binding constants were reported to be too large to be measured (61).
The increase in the organic phosphate affinity of chicken and
bar-headed goose Hbs over human Hb may be attributed to the
substitution of 3 basic residues found in the
subunits of most
avian Hbs. The replacement of Ala
135(H13),
Asn
139(H17), and His
143(H21) in human Hb
with Arg
135, His
139, and
Arg
143 in chicken Hb increases the positive charge of
the phosphate binding cavity (62).
82 and Arg
143 from one
subunit and
the side chains of Lys
82, His
139, and
Arg
143 from the second
subunit (Fig.
4A). The R- to T-state
transition opens up this cavity in most vertebrate Hbs. A deoxy model
of chicken Hb D based on human Hb·IHP permits IHP to enter the
phosphate-binding cavity. This position of IHP allows the side chains
of His
2, Lys
82, Arg
135,
His
139, and Arg
143 combined with the
amino termini of both
subunits to interact with five of the six
phosphate groups of IHP (Fig. 4B). These models suggest that
His-
139 (H17) and Arg
143(H21) might increase the IHP
affinity of both forms of avian Hb whereas Arg
135(H13)
increases the IHP affinity of only the deoxy form.
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Fig. 4.
A computer model of IHP binding to the
R-state (top) and T-state (bottom) of
chicken Hb D. The T-state model of the chicken Hb D·IHP complex
is based on the human deoxy-Hb·IHP complex (62). The R-state Hb
D·IHP complex consists of the oxy-Hb structure with IHP moved 4.5 Å away from its T-state binding site. Note that the side chains of
His2 and Arg135 are not positioned to interact
with IHP in the R-state model whereas these residues can interact with
the organic phosphate effector in the T-state model.
55% saturation) points from each curve (Fig.
5). The slope of the line gives
n, the Hill coefficient. In all oxygen binding curves of Hb
D, the lower part of the curve is described by Hill coefficients that
vary between 1.5 and 1.7. The Hill coefficient of the upper part of its
O2-binding curve is also about 1.8 in the absence of all
organic phosphate effectors (Hb D alone). The addition of 2 mM IHP to Hb D increases the Hill coefficient of the upper
half of the oxygen-binding curve to 4.2 (Fig. 5A),
demonstrating that super-cooperativity does occur in vitro.
Unexpectedly, the Hill plots of chicken Hb A behaved similarly. The
upper half of Hill plots for the oxygen binding of Hb A showed elevated
levels of cooperativity in the presence of 2 mM IHP whereas the lower half of the plot showed Hill coefficients of 1.6 (Fig. 5B). The lack of super-cooperativity observed for chicken Hb
D in the absence of IHP is probably due to the low (102 µM heme) concentrations of Hb used in these experiments.
Super-cooperativity of chicken Hb D would not be observed at this
concentration because over 69% of deoxy-Hb D would exist as tetramers
given the tetramer-octamer association constant of 1.26 × 104 M
1 measured by sedimentation
velocity measurements (20). However, over 92% of deoxy-Hb D would
associate into octamers at Hb concentrations (~300 mg/ml) normally
found in chicken red blood cells. The observation of enhanced
cooperativity in chicken Hb samples with IHP suggests that this process
is linked to the binding of inositol pentaphosphate in vivo.
This result also implies that IHP aids the formation of
(
D2
2)2 octamers.
The increased stability of the tetramer-tetramer interaction would
allow the (
2
2)2 to be
maintained in partially ligated structures, thereby promoting
super-cooperativity. Additional work will be required to measure the
effect of IHP on the tetramer to octamer association constant.
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Fig. 5.
Hill plots of the oxygen-binding data
reported by Isaacks et al. (54). The plot of the
Hb D data (A) in the absence of IHP ( ) gives Hill
coefficients of 1.8 over a wide range of oxygen saturation. In
contrast, the upper region of the Hill plot for samples of Hb D in the
presence of IHP (
) is described by a Hill coefficient of 4.2, indicating that IHP binding is linked to the super-cooperativity
observed in chicken blood. Hill plots for the oxygen-binding curves of
chicken Hb A (B) indicate that this component also exhibits
super-cooperativity in the presence of IHP (
).
D subunits of the two
interacting tetramers. This model is based on three surface residues,
Lys
71(EF1), Gln
78(EF8), and
Glu
82(F3) which are conserved in the
D
chains but not in the
A chains (20). Despite the fact
that in the absence of IHP, only the Hb D component self-associates
(20), the observation of super-cooperativity in both chicken Hbs
suggest that the proposed model of the
D-
D interface may be incorrect. However,
the sedimentation experiments of Cobb et al. (20) indicate
that the
chains do influence the tetramer-tetramer interaction. The
correct model of the tetramer-tetramer interface will require the
crystallographic analysis of the T-states of either Hb A or Hb D. Crystals of what should be deoxy-Hb D were obtained that diffract to
6.0-Å resolution when exposed to x-rays produced by a rotating anode
generator. Unfortunately, the resulting diffraction pattern could not
be indexed due to the presence of crystal plating, and the evidence of
1 unit cell axis which is greater than 300 Å. Nevertheless, a
significant amount of information has been inferred from the structural
analysis of the R-state of Hb D.
and
chains of Hbs A and D from birds and
Hb A from humans revealed that 8 of these residues are retained in many
avian Hbs. Three of these residues (Tyr
89,
Leu
10, and Met
59) have small aliphatic or
hydrophilic counterparts in human Hb A, suggesting that
Tyr
89, Leu
10, or Met
59
might participate in tetramer-tetramer association. A large, hydrophobic residue (either a phenylalanine or tyrosine) is present at
89 in all 20
D chains, but is replaced with either a
glutamine or histidine in most avian and mammalian
A
chains, respectively. It seems less likely that this residue directly
contributes to the association process because both chicken Hbs A and D
exhibit super-cooperativity in the presence of IHP. The other two
hydrophobic surface residues, Leu
10(A7) and
Met
59(E3), are found in all but three avian Hbs.
Leu
10 and Met
59 are replaced in human Hb
by an alanine and lysine, respectively. It is well known that the
replacement of a single polar residue by a hydrophobic residue in
sickle cell anemia can cause human Hb to polymerize (70). Therefore, it
is possible that the tetramer-tetramer interface involves one or more
of these hydrophobic surface residues.
The hydrophobic residues located on the surface of chicken oxy-Hb D
dimers onto the corresponding subunits of human deoxy-Hb. The
computer program GRASP (51) was used to calculate the surface
potentials of the deoxy-Hb D and human deoxy-Hb structures. The most
distinctive feature of the surface of chicken Hb D is a large patch of
positive charge that corresponds to the IHP-binding site located
between the two
subunits (Fig.
6A). A similar positive patch
is seen in human Hb (Fig. 6C), but the positive charge is clearly less intense. The increased positive charge allows chicken Hbs
to have a greater affinity for organic phosphates than human Hb A as
discussed above.
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Fig. 6.
Two views of the electrostatic surfaces of a
computer model of deoxy-Hb D. A and B,
compared with that of the human Hb structure (C and
D). Views shown are looking into the organic
phosphate-binding site (A and C) and looking at
the oval-shaped protrusion formed by the D and E helices (B
and D). The regions of each surface with negative charge, no
charge, and positive charge are shown in red,
white, and blue, respectively. Compared with
human Hb, the surface of the chicken Hb model has a greater number of
positively charged residues near its organic phosphate-binding site and
a lack of charge present on the surface formed by the D and E
helices.
subunits from the chicken Hb D model (residues 45-59) is also significantly different from that of human Hb. This surface in the
chicken deoxy-Hb D model appears as an uncharged, oval-shaped protrusion, surrounded by patches of charged residues (Fig.
6B). An apolar patch is also present on the surface of Hb A
of the bar-headed goose. Unfortunately, it is not known if bar-headed goose Hb exhibits super-cooperativity in the presence of IHP or if
goose Hb A self-associates. The same region in human Hb has a similar
shape, but includes several charged residues (Fig. 6D). The
hydrophobic surface formed by residues 45-59 of the chicken
subunits is the region most likely to participate in contacts between
two deoxy-Hb tetramers. An interface involving these residues also
makes good chemical sense because this interface would include Leu
55(D6) and Met
59(E3), two hydrophobic
residues which would be expected to contribute favorably to the
formation of an interface between two Hb D tetramers. Additional
contacts may also be involved.
2
2 tetramers. Lamprey Hb also exhibits a
deoxygenation-linked self-association (71-74) which involves
interactions between E
helices.2 The cooperative
mechanism of chicken Hbs could be similar to that observed in Lamprey
Hb2 because the proposed hydrophobic interface is formed by
parts of the D and E helices and is located close to the distal
histidine. We propose that formation of the tetramer-tetramer interface
in chicken Hbs mediates a shift in the E helix of a
subunit in such
a way that the distal histidine is pushed further into the heme pocket
as it is in lamprey deoxy-Hb.2 The resulting position of
the distal histidine would hinder oxygen binding, thereby reducing the
oxygen affinity of this subunit. The binding of oxygen to one of the
subunits would require that the distal histidine move back to an
R-state position, thus disrupting the tetramer-tetramer interface and
producing
2
2 tetramers of increased
oxygen affinity. Experiments are now underway to test this model by
crystallizing the deoxy forms of chicken Hbs A and D to define the
tetramer-tetramer interface that gives rise to super-cooperativity.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Elisabeth Karpova for crystallization of deoxy-Hb D and Dr. Diane McCarthy for helpful comments on the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by Texas Advanced Research Grant 003658-189 (to M. L. H.), Robert A. Welch foundation Grant 1219 (to M. L. H.), and National Science Foundation Grant MCB-9723825 (to A. F. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors of Hb D (code 1hbr) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed: The University of Texas at Austin, Austin, TX 78712. Tel.: 512-471-1105; Fax: 512-471-8696; E-mail: m.hackert{at}mail.utexas.edu.
2 H. Heaslet and W. E. Royer, private communication.
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ABBREVIATIONS |
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The abbreviations used are: Hb, hemoglobin; IHP, inositol hexa- phosphate; NCS, noncrystallographic symmetry; r.m.s., root mean square.
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REFERENCES |
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