From the Department of Physiology and Biophysics,
Albert Einstein College of Medicine, Bronx, New York 10461, the
** Department of Medicine, Division of Hematology, Albert
Einstein College of Medicine, Bronx, New York 10461, the
Department of Anatomy and Structural
Biology, Albert Einstein College of Medicine, Bronx, New York 10461, and ¶ INSERM, Unité 473, 84 rue du Général
Leclerc, 94276 Le Kremlin-Bicêtre, France
Received for publication, January 22, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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The impact upon molecular structure of
an additional point mutation adjacent to the existing E6V mutation in
sickle cell hemoglobin was probed spectroscopically. The UV
resonance Raman results show that the conformational consequences of
mutating the salt bridge pair,
Sickle cell hemoglobin (Hb S,1 Lesecq et al. (1, 2) have been investigating whether
modification of the polarity close to the Glu7-
Lys132, are dependent on
which residue of the pair is modified. The
K132A mutants exhibit the
spectroscopic signatures of the R
T state transition in both
the "hinge" and "switch" regions of the
1
2 interface. Both singly and doubly
mutated hemoglobin (Hb)
E7A exhibit the switch region
signature for the R
T quaternary state transition but not the hinge
signature. The absence of this hinge region-associated quaternary
change is the likely origin of the observed increased oxygen binding
affinity for the Hb
E7A mutants. The observed large decrease in
the W3
14
15 band intensity for doubly mutated Hb
E7A is
attributed to an enhanced separation in the A helix-E helix tertiary
contact of the
subunits. The results for the Hb A
Glu7-
Lys132 salt bridge mutants
demonstrate that attaining the T state conformation at the hinge region
of the
1
2 dimer interface can be achieved through different intraglobin pathways; these pathways are subject to
subtle mutagenic manipulation at sites well removed from the dimer interface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
6(A3) Glu
Val) exhibits the property of
anomalous and pathologic self-assembly. DeoxyHb S forms polymers in the
erythrocyte, which leads to microvascular blockage, organ damage, and
often premature death. Structure-based drug design requires knowledge
of optimal polymer disruption sites. The specific interaction in the Hb
S polymer involves the steric fit of the mutated hydrophobic
6 donor
site in a hydrophobic acceptor site located in an adjacent Hb S
tetramer. It is well known that the hydrophobicity and the
stereospecificity of the donor site are essential to the initiation of
the polymerization.
6 site could influence the
packing of the donor and acceptor sites, thus modifying the polymerization process. Replacing the hydrophilic Glu
7(A4) residue with a hydrophobic Ala residue resulted in a decreased polymerization of the doubly mutated rHb
E6V/E7A. It was postulated (1, 2) that the loss of the normal salt bridge between
Glu7(A4)
and
Lys132(H10) in the rHb
E7A mutants might lead to
an alteration in both the position and the mobility of the A helix,
illustrated in Fig. 1. These alterations
of the A helix might result in a misfit between the donor and acceptor
sites, which could explain the observed diminution in polymerization.
It follows from this hypothesis that modifying the other partner of the
salt bridge,
Lys132(H10), should have similar
consequences on polymerization (2).
View larger version (16K):
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Fig. 1.
Schematic representation of the effects of
the combination of E6V and
E7A mutations on the conformation of the Hb helices
A, E, and H. The deoxygenated quaternary structure for Hb S was
taken from the Protein Data Bank (structure number 2HBS). The
E7A
mutation was introduced into the structure using the Hyperchem software
program. Top panel, Hb
E6V. The E7-K132 salt bridge is
intact, and the A helix W15-S72 hydrogen bond is present. Bottom
panel, the additional E7A mutation breaks the salt bridge,
resulting in a separation of the A and E helices.
Visible resonance Raman spectroscopy is very useful in providing
detailed information relating to the influence of tertiary and
quaternary structure upon specific heme-related vibrational degrees of
freedom (3-7). Additionally, UV resonance Raman spectroscopy (UVRR)
provides information about vibrational modes of aromatic residues
within the globin. UVRR studies of hemoglobin from several research
groups have shown a consistent pattern of tertiary and quaternary
structure shifts coupled to spectral changes in specific tyrosine and
tryptophan bands (8-17). In particular and most significantly, spectral features have been clearly identified that reflect the key
determinants of the quaternary state, specifically in the "hinge"
(Trp37) and "switch" (
Tyr42) regions
of the
1
2 interface. Band intensities
also respond to the packing of the A helix against the E helix and to
the integrity of the salt bridge-derived scaffolding that maintains
interhelical separations, a feature that is indicative of different
tertiary structures within a given quaternary structure. Thus, the UVRR technique allows us to couple modifications of both local and global
elements of structure with observed functional changes. For example, Hb
C (
6(A3) Glu
Lys) is yet another naturally occurring mutant of
Hb A, which forms crystals in erythrocytes. UVRR spectroscopy, in
conjunction with other spectroscopic techniques, has been used to show
that the effect of the
6 mutation is communicated to both the A
helices and the central cavity where effectors such as inositol
hexaphosphate (IHP) bind (18-20).
In this study, UVRR spectroscopy is utilized to probe the
conformational consequences of disrupting the salt bridge between Glu7(A4) and
Lys132(H10) through
examination of the tryptophan W3 and tyrosine Y8 bands, which are
reporter bands for Hb tertiary and quaternary structure. The results
for singly and doubly mutated recombinant Hbs
E6V,
E7A,
K132A,
E6V/E7A, and
E6V/K132A are compared with those for both wild type
and recombinant Hb A (Hb A and rHb A, respectively). The deoxygenated
and CO derivatives of each Hb species are examined, as is the CO
derivative in the presence of IHP. The UVRR results reveal the impact
of the
Glu7-
Lys132 salt bridge mutations
on the positioning of the A helix and on the functioning of the
quaternary
37 hinge, which suggest a molecular explanation for the
macroscopic changes in polymerization and ligand binding for these mutants.
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MATERIALS AND METHODS |
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Preparation of the Recombinant Hbs--
The E6V,
E7A, and
K132A mutations were introduced into the
-globin cDNA by
site-directed mutagenesis using synthetic primers (Genset, France). The
mutated
-globin subunits were produced as fusion proteins in
Escherichia coli, using the expression vector pATPrcIIFX
-globin (21). After extraction and purification, the
fusion proteins were cleaved by digestion with bovine coagulation factor Xa (22). The presence of the mutation(s) was confirmed by
reverse-phase high performance liquid chromatography of the tryptic
digests and amino acid analysis of the abnormal peptides. The purified
-subunits were folded in the presence of cyanhemin and the partner
-subunits prepared from wild type Hb A, to form the tetrameric Hb
2
2 (21, 23). The folded recombinant
tetrameric Hbs were purified by preparative isoelectrofocusing on
Ultrodex dextran gel using Ampholine (Amersham Biosciences, Uppsala,
Sweden). Electrophoretic studies included electrophoresis on cellulose acetate and analytical isoelectrofocusing of the recombinant Hbs.
UVRR Spectroscopy--
The Hb samples were all at a
concentration of 0.5 mM heme in 50 mM Hepes at
pH 7.35. 0.4 M sodium selenate was added as an internal
standard, yielding a UVRR band at 834 cm1. Where
applicable, the IHP was 0.75 mM or six times the Hb
tetrameric concentration. The data were collected on samples chilled to
10 ± 4 °C to minimize photodamage. An argon laser system,
described elsewhere (20), was used to generate the excitation
wavelength of 228.9 nm with an incident laser power of 1.8 mW. Four
3-min acquisitions were accumulated for each ligation state of each Hb
variant over a 820-1670 cm
1 frequency window. The UVRR
data are averages of these four independent measurements. The
measurements for the CO-ligated HbE6V/K132A with and without IHP were
accumulated rather than averaged; the error bars are thus not shown in
Fig. 4 for these species. The data frequency scale was calibrated with
indene and toluene and is accurate to ±1 cm
1. The issue
of spectral reproducibility was addressed in the following manner: 1)
The absorption spectra were collected before and after exposure to the
UV laser beam. 2) If absorption changes were noted, the sequential UVRR
acquisitions were examined for evidence of band changes. 3)
Acquisitions that showed substantial changes were rejected and not
included in the final UVRR average spectra. That is, separate
acquisitions that were consistent in peak intensity and frequency as
well as band shape were included in the averaged spectra. Spectral data
were truncated to a 1530-1650 cm
1 frequency window, and
the intensity was normalized at the W3 band (~1558 cm
1)
for each set of spectra in Figs. 2 and 3. The software program Grams/32
AI, version 6.00 (Galactic Industries Corp., Salem, NH) was used to
determine the Y8a and W3(
14
15) peak heights used in Table I (full
width at half-maximum) using the subroutine, Peakstat, and for curve
fits to the W3 band, from which band heights for the
37 shoulder at
~1548 cm
1 were determined. All W3 bands were fit to two
curves of 0.7 Lorentzian/0.3 Gaussian band shape as the vibrational
signature for the
14 and
15 Trps is coincidental and cannot be
resolved; the second curve is for the
37 Trp. All of the band
intensities given in Table I and Fig. 4 were normalized against the
selenate 834 cm
1 peak. The numerical errors listed in
Table I and the error bars shown in Fig. 4 were determined
from the normalized peak intensities of the independent component
acquisitions used for each averaged UVRR spectrum as measured by the
aforementioned Peakstat subroutine. Common spectral processing
techniques include smoothing to improve the signal-to-noise ratio (24);
spectral smoothing, however, was not employed here.
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RESULTS AND DISCUSSION |
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Hb A
The changes in Hb globin structure that accompany ligation at the
heme can be followed by UVRR spectroscopy because of the critical sites
occupied by several of the UV-resonating Trp and Tyr residues. Two of
these are highly responsive to quaternary structural changes:
Trp37 and
Tyr42 are located in the hinge
and switch regions of the
1
2 dimer interface, respectively. In addition, there is a tryptophan on the A
helix of both the
(
14) and
(
15) subunits that provides a
UVRR signature for the status of the packing distance between the A and
E helices (25). For human wild type Hb A, the change in ferrous heme
ligation state from fully ligated to fully deligated (deoxy) is
accompanied by the R
T state quaternary structure transition. The
conformational changes of the aromatic residues in the interface
following the R
T state transition yield two major UVRR spectral
changes. These are a ~2-cm
1 increase in frequency of
the Y8a band from ~1615 to ~1617 cm
1 and an intensity
increase of ~37% in the W3 shoulder (W3
37) at 1549 cm
1 (7, 9). Several investigators have used these UVRR
spectral changes to characterize the effect of site mutation on
hemoglobin (6, 7, 9, 10, 12-15, 17, 20, 25-28). Thus, the association of these small spectral changes with specific changes in hemoglobin structure and ligation state is both well documented and consistent. The UVRR spectra shown in Figs. 2 and
3 have been truncated to highlight the
1530-1650 cm
1 frequency window. This spectral window
contains the conformation-sensitive W3 and Y8a reporter bands.
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The W3 band at ~1558 cm1 has two contributions (9, 13,
27, 29). The central feature that peaks at ~1558 cm
1 is
derived from the two A helix tryptophans (
14 and
15), whereas the R
T state sensitive shoulder at ~1550
cm
1 originates from
Trp37. Intensity
changes in the central peak of the W3 band at ~1558 cm
1
have been shown to originate from variations in the hydrogen bond
between the A helix tryptophans and their hydrogen-bonding partners on
the E helix (25). Increased and decreased intensities correlate with
increases and decreases in hydrogen bond strength, respectively.
Modulation of the hydrogen bond strength is attributed to changes in
the distance between the two helices.
The intensity for the ~1550 cm1 shoulder on
the W3 band increases as the hinge region of the
1
2 interface undergoes an R
T state
transition, reflecting the changes in the hydrogen bonding pattern of
Trp37 with
Asp94. Thus, the W3 band
provides information on both the functionally important hinge region of
the
1
2 interface and the packing of the A
and E helices. The tyrosine-derived Y8a band at ~1616
cm
1 shows a ~1-2-cm
1 shift to higher
frequency when liganded R-state Hb A is converted to the deoxy T state
(9, 29). This frequency shift originates primarily from
Tyr42 (13, 17, 28) in the switch region of the
1
2 interface. The intensity of this band
has been correlated with the integrity of the scaffolding supporting
the H helices.
The intensity of the UVRR spectra in each set in Figs. 2 and 3 have
been normalized at the W3 peak to clarify the differences in the 37
shoulder at 1548 cm
1. Both wild type Hb A and rHb A (Fig.
2, top panel, sets a and b,
respectively) yield the well documented Y8a frequency increase (2.9 and
1.8 cm
1, respectively; Table
I), and the W3
37 band intensity
increase (0.43 and 0.44, respectively; Table I) associated with the R
T state transition, as discussed above. The spectral
difference between the W3
37 bands for the two hemoglobin
ligation states is 1 order of magnitude above the noise level of the
constituent spectral acquisitions. For both species of Hb A, ligation
results in an increase of intensity for the W3
14
15 band (Fig.
4a and Table I), whereas the
Y8a band intensity increases for ligated wild type Hb A only (Fig.
4b and Table I). Addition of a 5-fold excess of the
effector, IHP, to ligated forms of both Hb As results in an enhancement
of the W3
37 band intensity, which is indicative of a more T
state-like hinge region, but apparently has little or no effect on the
Y8a band position, which remains R state like (Fig. 2, top
panel, sets a and b, and Table I). The
addition of IHP has a large enhancement effect on the Y8a band
intensities of both ligated Hb As (Fig. 4b and Table I) and
on the W3
14
15 band intensity of wild type Hb A (Fig.
4a and Table I).
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Hb S (E6V)
The upshift in the Y8 band frequency and the W3 37 band
intensity increase accompanying the R
T state transition for both Hb As are also found in the UVRR results for wild type Hb S and rHb S
(Fig. 2, bottom panel, sets a and b,
respectively, and Table I). The W3
37 band intensity increase for
deoxy wild type Hb S is comparable (0.45) with that obtained for either
species of deoxy Hb A (0.43-0.44), whereas the intensity increase
obtained for deoxy rHb S is higher (0.53) (Table I). Conversely,
ligation, which corresponds to the T
R state transition, results in
intensity increases for both the W3 and Y8 bands of wild type Hb S
(0.19 and 0.26, respectively; Table I) and for those of rHb S (0.13 for
W3 and 0.17 for Y8a; Fig. 4 and Table I). Addition of IHP to wild type
COHb S yields little, if any, decrease in the W3 band intensity (Fig.
4a and Table I) and an unchanged Y8 band intensity (Fig.
4b and Table I). For COrHb S, the addition of IHP results in
an increase in the Y8a band (Fig. 4b and Table I).
rHb E7A
The characteristic Y8a frequency upshift accompanying the R T
state transition is also manifest by rHb
E7A (Fig. 3, top panel, set a, and Table I). The usual W3
37
intensity increase, however, is not found in the UVRR results for this
rHb mutant (Fig. 3, top panel, set a, and Table
I). The W3
14
15 and Y8a band intensities for deoxy rHb
E7A are
similar in magnitude to those of wild type deoxyHb A (Fig. 2, top
panel, set a), whereas ligation to rHb
E7A yields a
small decrease in the W3
14
15 band intensity and no change in the
Y8a band intensity (Fig. 4 and Table I). The addition of IHP to COrHb
E7A leads to intensity increases in the Y8a and W3
37 bands: a
0.36 increase in Y8a relative to the Y8a band of deoxy rHb
E7A (Table
I) and a 0.16 increase in the W3
37 band, comparable with the 0.11 band increase found in the results for wild type COHb A + IHP (Table
I). Under the same conditions, the W3
14
15 band intensity has
merely been restored to its deoxy value (Fig. 4a and Table
I).
rHb E6V/E7A
The UVRR results for rHb E6V/E7A following the R
T state
transition are similar to those for rHb
E7A; the Y8a frequency upshift occurs, but the W3
37 band intensity increase does not (Fig.
3, top panel, set b, and Table I). This double
mutation results in the lowest deoxy W3
14
15 and Y8a band
intensities observed for this set of Hbs (Fig. 4). Ligation
substantially increases the intensities of both bands (Fig. 4 and Table
I). Addition of IHP to the ligated species has a small effect on the intensity of the W3
14
15 band (Fig. 4a) but results in
a substantially enhanced W3
37 band intensity (Fig. 3, top
panel, set b, and Table I). The intensity of the Y8a
band is minimally influenced by the addition of IHP to CO rHb
E6V/E7A (Fig. 4b and Table I).
rHb K132A
The K132A mutation did not affect the Y8 band shift associated
with the R T state transition (Fig. 3, bottom panel,
set a, and Table I). The W3
37 band intensity increase
associated with this transition, however, was greater than that seen
for wild type HbA (Fig. 3, bottom panel, set a,
and Table I). Both the W3
14
15 and Y8a band intensities for deoxy
rHb K132A are higher than those for wild type deoxyHb A (Fig. 4).
Ligation to rHb K132A further enhances both of these bands, and the
addition of IHP leads to additional band intensity increases, with a
nearly 2-fold Y8a band increase for CO rHb K132A + IHP over that of
wild type deoxyHbA (Fig. 4 and Table I). In contrast to the results for
the ligated rHb mutants discussed above, the addition of IHP to CO rHb
K132A does not result in an increase in the W3
37 band (Fig. 3,
bottom panel, set b, and Table I).
rHb E6V/K132A
The UVRR results for the R T state transition of rHb
E6V/K132A are similar to those for rHb K132A (Fig. 3, bottom
panel, and Table I). Generally, the W3
14
15 and Y8a band
intensities for deoxy rHb
E6V/K132A are greater than those observed
for wild type deoxyHbA (Fig. 4). Ligation, however, has little effect
on either the W3
14
15 band intensity or on the intensity of the Y8a band (Fig. 4). The addition of IHP enhances both the W3
14
15 and Y8a bands, but the Y8a band for ligated rHb
E6V/K132A is reduced
relative to that for ligated rHb K132A (Fig. 4b). As for CO
rHb K132A, the addition of IHP does not enhance the intensity of the W3
37 band (Fig. 3, bottom panel, set b, and
Table I).
Two categories of UVRR spectral differences are observed in comparing
the different Hb derivatives. One set is associated with R T
state differences and is comprised of a Y8a band shift and a W3
37
intensity increase. The former change is observed when comparing the
deoxy and CO derivatives for all the Hb species examined in the present
study, whereas the latter is observed for all species except
the E7A mutants. The second set of spectroscopic changes consists of
intensity changes in the W3
14
15 and Y8a UVRR bands when
comparing both the different derivatives (ligation state) of a given Hb
and the same derivative from different Hbs.
None of the Hb S-related mutations discussed here eliminate the Y8 band
1.5-3 cm1 upshift (Figs. 2 and 3 and Table I) associated
with the switch motion of the
Tyr42(C7) during the R
T state transition. This upshift has been shown to originate largely
from
Asp99(G9) hydrogen bond donation to
Tyr42(C7) upon Hb ligation (9, 13, 30). Similarly, the
W3
37 band intensity increase, associated with the R
T state
transition hinge motion at the
1
2
interface (9), is seen in all the deoxy versus CO UVRR
comparisons for the Hbs discussed here except for those with
the E7A mutation (Figs. 2 and 3 and Table I). The W3
37 band
intensity increase has been associated with changes in the hydrogen
bond between
Trp37(C3) and
Asp94(G1).
The addition of IHP to the CO derivatives results in small to moderate
intensity increases for the W3 37 shoulder for the Hb A, the Hb S
species, and the E7A mutants but not for the K132A mutants (Figs. 2 and
3 and Table I). This intensity increase is in the direction of the R
T state transition-associated change. Several features are worthy
of additional comment. It is indeed intriguing that for the E7A
mutants, deoxygenation does not induce the typical R
T state
transition-associated intensity increase in the W3
37 band, but
addition of IHP to the liganded derivative does. This observation
indicates that there are clearly multiple intraglobin pathways for
inducing changes at the hinge region of the dimer interface. The
concept of multiple pathways is further substantiated by the results
from the E132A mutants, which exhibit the reverse effect in that
deligation but not the addition of IHP induces the R
T state
transition-associated changes in the hinge sensitive W3
37 shoulder.
The results also show that changes in the hinge and switch regions are
necessarily coupled because under the present conditions the addition
of IHP does not result in the Y8a, R
T state-associated frequency
shift. At the significantly lower pH of 6.3, IHP addition to the CO
derivatives of HbA, rHb
E6V, and rHb
E7A also induces the Y8 band
to partially upshift toward the T state value (data not shown).
R T state Transition-associated Spectral Changes
Effect of the E7A Mutation--
The rHb
E7A mutation
replaces a negatively charged residue with one that is noncharged and
aliphatic, resulting in the loss of the salt bridge with the positively
charged residue,
Lys132. Based on the W3
37 UVRR
results presented here, it appears that the disruptive effect of the
uncompensated
Lys132 charge on the hinge region of the
1
2 interface is equivalent to the loss of
the R
T state transition (Fig. 3, top panel). One
possible communication pathway for this disruption to the
Trp37 hinge region is through
Gln131,
which is adjacent to the
7 salt bridge partner,
Lys132, and is noncovalently linked to the interfacial
His103 (31). This disruption of the quaternary contacts
within the hinge region of the T state
1
2
interface is consistent with the increase in oxygen affinity of the rHb
E7A mutants reported by Lesecq et al. (1). Disruption of the
hinge via mutagenic manipulation of
37 has been shown to enhance the
ligand binding properties of the T state and reduce proximal strain at
the heme (32, 33).
Effect of the K132A Mutation--
The effect of eliminating the
7-
132 salt bridge by replacing positively charged
Lys132 with uncharged, aliphatic Ala is to enhance the R
T transition state-associated W3
37 band intensity increase
vis à vis that for wild type Hb A (Table I). Thus, the
loss of charge at
132 creates a "hyper" T state hinge signature.
This result, in association with those from the
E7A mutant,
indicates that uncompensated charge at the
132 site destabilizes the
T state hinge, whereas loss of charge at this site enhances the T state
hinge, and the function of the salt bridge is to modulate the T state
hinge by offsetting the charge at
132.
Y8a and W3 14
15 Band Intensity Changes--
The relative
intensities of the W3 and Y8 bands appear to follow a pattern upon
comparing derivatives of a given Hb. Although there are several
exceptions (Fig. 4), the intensity of the W3 and Y8 bands generally
increases in the sequence of deoxy:CO:CO + IHP.
Doubly Mutated rHb E6V/E7A--
As shown in Fig. 4
and Table I, the UVRR spectra obtained from deoxy rHb
E6V/E7A show
the most pronounced intensity decrease for both the W3
14
15 and
Y8 bands. Building on the findings of Spiro and co-workers (9, 12, 13,
25), this intensity decrease in the W3
14
15 peak can be explained
by a separation of the A helix from the E helix of the
chains
caused by the combined change in the polarity and hydrophobicity of
both the
6 and
7 residues, as illustrated in Fig. 1. It is not
clear whether changes in hydration induce slight changes in the
helix itself. The
14 and
15 tryptophan residues are not
distinguishable by UVRR spectroscopy, but given that the mutations
studied here are located on the
chain, all of the differences
observed on the W3
14
15 main band can be reasonably assigned to
Trp15 (14).
Trp15 is an A helix residue
situated in the crevice formed by the A and E helix. It normally forms
a hydrogen bond with
Ser72 of the E helix in both the
deoxy and oxy structure (Fig. 1, top panel). A decrease in
the intensity of the 1558-cm
1 band reveals a weakening of
this hydrogen bond. In a study of C terminally deleted Hb A, Wang and
Spiro (7) observed intensity increases in the W3
14
15 (A12) peak
of both deoxy and ligated Hb A, which they attribute to a collapse of
the A helix toward the E helix. This collapse is purported to originate
from the loss of the C-terminal anchor of the H helix. The plausible
claim was made that the H helix acts as a scaffold for the A helix, which keeps the A helix separated from the E helix (Fig. 1). A weakening of the scaffolding through changes in the hydrogen bonds and
salt bridges of the H helix allows the A helix to pack more tightly
against the E helix, thus allowing for a stronger
Trp15(A12) to
Ser72(E16) hydrogen bond.
Hirsch et al. (19) observed a decreased intensity of the W3
14
15 band of Hb C, where
6 glutamic acid is replaced by a
lysine residue. These authors demonstrated a weakening of the
Trp15(A12)-
Ser72(E16) hydrogen bond,
which is suggestive of a displacement of the A helix away from the E helix.
In the present study, the deoxy rHb E6V/E7A Y8a band is 16% less
intense than that observed for deoxy rHb S (Fig. 4b and Table I). Y8a band intensity changes can reasonably be attributed to
either or both of the two penultimate tyrosine residues in the Hb
molecule,
Tyr140 and
Tyr145. Both
residues are integral parts of the scaffolding linking the H and A
helices. The Y8a intensity changes observed in the present study are
likely to originate from
Tyr145.
Tyr145(HC2) occupies the pocket made by the H and F
helices in deoxy HbA and deoxy Hb S and forms a hydrogen bond with
Val98(FG5), which contributes to the scaffolding of the
A helix by the H helix. The decrease in the deoxy rHb
E6V/E7A Y8a
band intensity (Fig. 4b) is postulated to arise from a
weakening of the
Tyr145-
Val98 hydrogen
bond through a shift in the H helix.
Single Mutants, rHb E7A and
K132A and Doubly Mutated rHb
E6V/K132A: Involvement and Role of the Salt Bridge
7--
132
The W3 and Y8a bands intensities for deoxy rHb
E7A are similar to those of deoxy rHb S (Fig 4 and Table I). No
weakening of the hydrogen bond involving
Trp15(A12) and
Ser72(E16) can be inferred. This result suggests that
the absence of the salt bridge between
Glu7(A4) and
Lys132(H10) per se does not influence the
separation of the A and E helices and the A and H helices.
This claim is supported by the results from mutants rHb K132A and
rHb
E6V/K132A. The W3 and Y8a bands for the deoxy derivative of
these rHbs exhibit increased intensities compared with that for deoxy
rHb S (Fig. 4 and Table I). In these mutants, the
7 (A4)-
132
(H10) salt bridge is also absent, whereas the
Trp15(A12)-
Ser72(E16) hydrogen bond is
apparently strengthened, showing a tighter packing of the A helix
against the E helix as follows from the discussion above. The other
ligation state derivatives of these mutants show either no additional
change or a further increase in the W3 and Y8a bands intensities (Fig.
4 and Table I). It appears that complete loss of the charge at position
132 results in a loss of the A-H scaffolding with a concomitant
decrease in the spacing between the A and E helices. It follows that
the
7-
132 salt bridge modulates the charge at the
132 residue
in a way that allows for the appropriate degree of interhelical
scaffolding and proper behavior of the hinge region at the dimer interface.
Importance of the Hydrogen Bond Involving Tyr145 and
Val98--
In a study of
Tyr145 mutants,
Ishimori et al. (34) showed that the presence of the
phenolic side chain in the H-F pocket is a contributing factor to T
state stability, whereas the hydrogen bond strength between
Tyr145(HC2) and
Val98(FG5) is a
modulating factor for the extent of proximal strain within the T state.
Loss of the hydrogen bond is associated with a decrease in proximal
strain as reflected in an increase in the frequency of the
iron-proximal histidine-stretching mode for the deoxy derivative of the
Y145F mutant. A subsequent study by Togi and co-workers (35) showed
that this hydrogen bond is also important in stabilizing the R
T
transition state. Loss of the hydrogen bond increased the energy of the
transition state and thereby slowed the R
T state transition.
The current study suggests that the A helix mutations E6V and E7A lead
to modifications of the scaffolding through changes in hydrogen bonds
and salt bridges to the H helix. The decrease in Y8a UVRR band
intensity observed for the deoxy rHb E7A mutants may indicate a
weakening of the
Tyr145(HC2)-
Asp98(FG5)
hydrogen bond because of the aforementioned modifications. Based on the
145 mutant studies described above, this proposed bond weakening
should result in a higher oxygen affinity for Hb, which has been
observed for singly and doubly mutated Hb
E7As (1).
Conclusion
The spectroscopic results in this study show that the consequences
of disrupting the salt bridge between Glu6 and
Lys132 is a function of which contributing residue is
altered. The salt bridge clearly does not function simply as a taut
spring linking two helices together, which when disrupted results in an
automatic separation of the spring-linked elements. The salt bridge
appears to modulate the effect of the charge of
Lys132.
Complete loss of the charge, as occurs in the
K132A mutants, results
in spectroscopic signatures for a collapse of the scaffolding supporting the A and H helices leading to a more "compressed" overall structure. The resulting structure is still capable of undergoing the full range of R
T state transition-associated hinge
and switch motions as reflected in the UVRR spectrum. There are also
spectroscopic indications that the charge at
132 effects the
stability of the T state hinge region of the
1
2 dimer interface. Both the single and
double
E7A mutants, where the charge on
132 is fully
unshielded, fail to show the hinge region-associated W3
37 band
intensity increase for the R
T state transition that is seen for
all the other mutants, including
K132A. The absence of the R
T
state change in the hinge region is a likely factor in the increased
oxygen binding affinity observed for the rHb
E7A mutants. Thus,
too little shielding of the charge at
Lys132 results in
an altered T state hinge region of the
1
2
dimer interface, whereas a complete neutralization of the charge
results in a compaction of the overall structure. The
Glu7-
Lys132 salt bridge appears to play
a role in supporting the appropriate charge balance that in turn
maintains the A helix-H helix scaffolding and the proper T state hinge.
The UVRR spectrum from the double mutant Hb E6V/E7A indicates an
enhanced separation in the A helix-E helix tertiary contact, as
reflected in a weakening of the hydrogen bond between
Trp15 and
Ser72. This finding supports
the idea that a change in the A helix packing is responsible for the
observed decrease in Hb
E6V/E7A polymerization (1, 2). It is not
clear whether the weakening of the hydrogen bond between
Trp15 and
Ser72 is purely the result of
the local perturbation on the A helix or the combined result of the A
helix mutations with the unshielded charge at
Lys132.
The double mutant, Hb
E6V/E7A, also shows a substantially
decreased Y8a band intensity, attributed to an increased separation
between the H helix and the F helix. The combined effect is suggestive of a global expansion or loosening of the tertiary structure.
The spectroscopic changes occurring upon addition of IHP to the
CO-saturated derivatives of all the species examined is consistent with
IHP inducing a general tightening of the overall globin structure. This
tightening is reflected in spectroscopic signatures of a strengthened
hydrogen bonding between the A and E helices and the H and F helices.
At pH 7.35, addition of IHP to the CO saturated derivatives does not
perturb the switch region of the 1
2 dimer interface but does induce T state character into the hinge region of
all but the
K132A mutants. Thus, the
E7A mutation eliminates the
deligation-induced R
T state hinge transition but not the IHP-induced effect on the hinge, whereas the
K132A mutation
eliminates the IHP effect but maintains (or even enhances) the
deligation-induced change. It follows that attaining the T state
conformation at the hinge region of the
1
2 dimer interface can be achieved
through different pathways and that these pathways are subject to
subtle mutagenic manipulation at sites well removed from the interface.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants PO1 GM58890 and R01 HL58247, American Heart Association Heritage Affiliate Grant 9950989T, and Institut National de la Santé et de la Recherche Médicale and the Association Recherche et Transfusion Contract 21-2000.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.
We dedicate this paper to the memory of Josée Pagnier, colleague and friend. Fortunately, she was able to see this work completed. She remains in our memory as a long standing collaborator. One of us (R. L. N.) was involved in her thesis work and remembers her with affection and gratitude in the pursuit (with Dominique Labie) of the origin of the sickle gene in Africa.
§ These authors contributed equally to this work.
Supported by the Délégation Génerale pour
l'Armement (Ministère de la Défense).
§§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461. Tel.: 718-430-3591; Fax: 718-430-8819; E-mail: jfriedma@aecom.yu.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M200691200
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ABBREVIATIONS |
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The abbreviations used are: Hb, hemoglobin; Hb S, sickle cell hemoglobin; Hb A, wild type human hemoglobin A; rHb, recombinant Hb; IHP, inositol hexaphosphate; UVRR, ultraviolet resonance Raman.
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