(Received for publication, December 18, 1996, and in revised form, April 7, 1997)
From INSERM, Unité 299, Hôpital de Bicêtre, 94275 Le Kremlin-Bicêtre, France
Hb S variants containing Lys-132
Ala or
Asn substitutions were engineered to evaluate the consequences of the A
helix destabilization in the polymerization process. Previous studies
suggested that the loss of the Glu-
7-Lys-
132 salt bridge in the
recombinant Hb
E6V/E7A could be responsible for the destabilization
of the A helix. The recombinant Hb (rHb) S/
132 variants polymerized with an increased delay time as well as decreased maximum absorbance and Hb solubility values similar to that of Hb S. These data indicate that the strength of the donor-acceptor site interaction may be reduced
due to an altered conformation of the A helix. The question arises
whether this alteration leads to a true inhibition of the polymerization process or to qualitatively different polymers. The
oxygen affinity of the
132 mutated rHbs was similar to that of Hb A
and S, whereas the cooperativity and effects of organic phosphates were
reduced. This could be attributed to modifications in the central
cavity due to loss of the positively charged lysine. Since Lys-
132
is involved in the stabilization of the
1-
1 interface, the loss
of the
132(H10)-
128(H6) salt bridge may be responsible for the
marked thermal instability of the
132 mutated rHbs.
The substitution of valine for the 6 glutamic acid residue in
human Hb results in the abnormally low solubility of deoxy-Hb S. Under
physiological conditions, sickle Hb aggregates upon deoxygenation to
form a gel composed of long helical fibers that deform the erythrocytes
and severely diminish their life-span. Electron microscopy (1, 2) and
x-ray crystallographic studies (3) have shown that both fibers and
crystals are composed of double strands of deoxy-Hb S molecules. The
formation of the double strands requires stereochemical contact between
complementary surfaces involving specifically Val-
6 and a
hydrophobic pocket on an adjacent molecule (lateral contact).
The capacity of some Hb variants to facilitate or impair the
polymerization process of Hb S is well documented (4). The use of
binary Hb mixtures (Hb X + Hb S) has allowed a mapping of the residues
involved in areas of contact in the polymer (5). The consequences of
mutations associated with Val-6 on the same
chain are less well
known. Six naturally occurring Hb variants have been described with two
mutations on the same
chain, one of them being the Hb S
substitution. Among them, Hb S Antilles Val-
6/Ile-23 (6) and Hb S
Oman Val-
6/Lys-121 (7) exhibit an increased propensity to form
polymers. Site-directed mutagenesis and expression in heterologous
systems allows determination of the contribution of various sites in
the polymerization process (8-10). Studies of recombinant Hbs
(rHbs)1 have shown that in Hb S Antilles
the polymer fibers were stabilized at the axial contact by the
replacement of Val with the more hydrophobic residue Ile (10). We have
previously reported studies of the function and polymerization of
another rHb,
E6V/E7A (11). In this rHb, the association of
Glu-
6(A3)
Val and Glu-
7(A4)
Ala mutations on the same
chain (rHb
E6V/E7A) results in an apparent decrease of the polymer
formation. We therefore postulated that this decrease could be due to
an instability of the A helix because of the loss of a salt bridge
between the A and H helices, namely between the Glu-
7(A4) and
Lys-
132(H10) residues (11). Modification of the second partner in
the salt bridge (Lys-
132(H10)) may also result in its rupture. This
residue participates in several contacts at the
1-
1 interface
(12) and might also be involved in the stability of the A helix. We
have thus engineered two doubly mutated Hbs in which the Glu-
6(A3)
Val mutation is associated with either Lys-
132(H10)
Ala or
Asn substitution (rHbs
E6V/K132A and
E6V/K132N, respectively). We
have also engineered the single mutant Hbs
Lys-
2
2132(H10)
Ala and
Lys-
2
2132(H10)
Asn as controls (rHbs
K132A and
K132N, respectively).
The E6V,
K132A, and
K132N mutations were introduced
into the
-globin cDNA by site-directed mutagenesis using
synthetic primers (Genset, Paris, France). The mutated
-globin
subunits were produced as fusion proteins in Escherichia
coli using the expression vector pATPrcIIFX
(13). After
extraction and purification, the fusion proteins were cleaved by
digestion with bovine coagulation factor Xa (14). 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
natural Hb A) to form the tetrameric Hb
2
2 (13-15).
Electrophoretic studies included electrophoresis on cellulose acetate and isoelectrofocusing of the recombinant Hbs. Fluorescence studies of the rHbs were performed at a concentration of 10 µM on a heme basis in 10 mM phosphate buffer, pH 7.0, using an SLM 8000 spectrofluorometer. Fluorescence spectra were measured in the region for tyrosine and tryptophan emission (for air-equilibrated samples). The heat stability of the Hbs was determined by incubating the recombinant and native Hbs at 65 °C in 10 mM phosphate buffer, pH 7.0, at 100 µM heme under 1 atm of CO or 1 atm of O2 (16).
The oxygen binding curves were recorded at 25 °C with a continuous method using the Hemox Analyzer system (TCS Medical Products, Huntington Valley, PA) (17). Bimolecular recombination of CO was studied after flash photolysis dissociation with 10-ns pulses at 532 nm. Detection was at 436 nm for samples equilibrated under 0.1 atm of CO (18). Measurements were made at different protein concentrations to study the concentration dependence of the ligand binding kinetics to estimate the dimer-tetramer equilibrium compared with natural Hb A (19). For concentrations above 30 µM (on a heme basis), 1-mm optical cuvettes were used. Below 30 µM, 4 × 10 mm cuvettes were used with detection along the 10-mm axis and dissociation at 90° along the 4-mm axis.
Kinetics of polymer formation for mutant deoxy-Hbs were carried out in 1.8 M potassium phosphate buffer, pH 7.2, for different Hb concentrations, following initiation by a temperature jump (0 to 30 °C). The solubility and kinetics of polymerization of the mutated Hb were compared with those of native Hb S. Under these conditions and after a characteristic lag period, the assembly of Hb S into fibers resulted in a cooperative increase in turbidity at 700 nm until a plateau was achieved. The Hb solubility (Csat) was determined by measuring the Hb concentration of the soluble phase after completion of the polymerization process (20, 21).
Molecular graphics models of the deoxy-Hbs E7A and
K132N were
performed starting from the crystallographic coordinates of the deoxy
structure of Hb A (file 3HHB, Protein Data Bank, Brookhaven National
Laboratory) reported by Fermi et al. (22). Minimization of
the potential energy in the mutant and normal Hbs was performed using
the CHARMmTM program (version 22) (23) with a Silicon
Graphics Indigo work station. Holding the rest of the structure
constant, the mutated residue was initially minimized by starting with
an additional high harmonic constant that was progressively decreased
to 0.
Reverse-phase high performance liquid chromatography of the
tryptic digest of the purified mutated fusion proteins K132A,
K132N,
E6V/K132A, or
E6V/K132N revealed the presence of one or
two abnormal peptides. Amino acid analyses of these peptides confirmed
the presence of the expected mutation(s). Reassembled tetramers showed
visible absorption spectra identical to those of native Hb A and S in
dilute solution and liganded forms. The fluorescence intensity is
sensitive to the small quantities of apoproteins. The intensity for the
rHbs was similar to that for natural Hb A, indicating a correct
reconstitution to the holoprotein form.
Cellulose acetate electrophoresis and
isoelectrofocusing (Fig. 1) of the purified rHbs
K132A,
K132N,
E6V/K132A, and
E6V/K132N showed a single band
migrating at isoelectric points of 6.85, 6.85, 7.10, and 7.10, respectively, relative to 6.95 and 7.20 for Hb A and Hb S and 7.40 for
the rHb
E6V/E7A. These values are higher than expected for the
replacement of the positively charged Lys with Ala or Asn. This may be
explained by the location of the
132 residue, which is at least
partially buried in the central cavity of the Hb tetramer.
Heat stability of the oxy and carboxy forms of the mutated rHbs was
compared with that of the natural Hbs A and S and that of the rHb
E6V/E7A. As shown in Fig. 2, we did not observe
significant differences between Hb A and S. The stability of the
132
mutants was dramatically decreased under the same conditions. The
doubly mutated rHbs
E6V/K132A and
E6V/K132N were the most
unstable, more so than the rHbs containing only the
132 mutation.
The rHbs
E7A and
E6V/E7A were the least unstable mutants (Fig.
2).
Functional Studies
The oxygen equilibrium curves (not shown)
showed that the oxygen affinity of the rHbs K132N and E6V/K132N was
similar to that of native Hb A. When Lys-132 was replaced with Ala
the oxygen affinity was slightly increased (Table I).
The cooperativity in ligand binding and the 2,3-diphosphoglycerate
effect were decreased for all mutants. Since it is known that the
oxygen binding properties of Hb S are similar to those of Hb A, the
functional abnormalities could be attributed to the presence of the
132 mutation. CO recombination kinetics for the rHbs (not shown)
were similar to those observed for Hb A, indicating that the mutant
rHbs retain allosteric function. At low protein concentration, loss of
the allosteric form is normally observed due to the increased fraction
of dimers. Results at 1 µM indicate that the rHbs do not
present a significant increase in the amount of dimers.
|
Polymerization of the rHbs E6V/K132A and
E6V/K132N in the deoxy
form was studied in vitro by the temperature jump method and
compared with that of the rHb
E6V/E7A and natural Hb S. Fig. 3 illustrates the variations of
A700 as a function of time after the
temperature jump. Under these conditions, the rHbs
K132A and K132N
did not polymerize at all concentrations studied (up to 2.0 g/liter).
The kinetic curves of polymerization of the doubly mutated Hbs were
sigmoidal, as was observed for native Hb S. The maximum absorbance at
700 nm was lower than for Hb S, whereas the delay time (
) was longer
at all concentrations studied (1.6-3.0 g/liter). A logarithmic plot of
the reciprocal of the delay time versus initial Hb
concentration (Fig. 4) showed straight lines shifted
toward the right for the rHbs
E6V/K132A and
E6V/K132N, indicating
longer delay times compared with Hb S and the rHb
E6V/E7A. Plotting
the log of the aggregation rate as a function of log C showed straight
lines with similar slopes for Hb S and rHb
E6V/E7A on the one hand
and for the rHbs
E6V/K132N and
E6V/K132A on the other (Fig.
5). The time required to reach maximum polymerization depends on Hb concentration. At equivalent initial concentration that
time was longer when the sickle mutation was associated with the
132
mutations than when associated with Glu-
7
Val. The Csat values for the double mutants were not
significantly different from those of natural Hb S and rHb
E6V/E7A
(Fig. 6). For the two double mutants, the aggregation
process was reversible in the presence of CO and in ice water.
We have previously demonstrated that the association of the E7A
and
E6V mutations on the same
chain leads to a decreased polymer
formation; the Glu-
7(A4) for Ala substitution in human Hb resulted
in heat instability and in an increased oxygen affinity of the rHbs
E7A and
E6V/E7A (11). In human Hbs A and S, Glu-
7(A4) forms an
intrachain salt bridge with Lys-
132(H10) in both R- and T-state
structures (24). The loss of this salt bridge may modify the
conformation of the A helix, which could account for the alteration of
the polymerization process. We attributed the increased oxygen affinity
of the rHbs
E7A and
E6V/E7A to an increased dissociation of the
tetramer into dimers demonstrated by the concentration dependence of
the ligand binding kinetics. In the present work we have studied the
consequences of modifications of Lys-
132(H10), which also
participates in the salt bridge. In contrast with the data obtained
with the rHbs
E7A and
E6V/E7A, CO rebinding to the
photodissociated
132 mutants did not reveal an increased
dissociation into dimers, and the rHbs modified at the
132(H10) site
did not exhibit high oxygen affinity (Table I). Note that naturally
occurring Hb Yamataga Lys-
132
Asn is described as having a
slightly decreased oxygen affinity (25). In human deoxy-Hb, the
132
residue interacts with the N-terminal
-chain residues Val-
1(NA1),
His-
2(NA2), and Leu-
3(NA3) (12). Two of these residues
participate in the 2,3-diphosphoglycerate binding. The loss of a
positive charge when the Lys-
132 is replaced by either Ala or Asn
may result in the destabilization of the contacts in the central
cavity. These structural modifications are able to prevent or modify
the binding of the allosteric effector.
Structural crystallographic studies revealed that in both the R- and
T-state Lys-132(H10) is not only linked to Glu-
7(A4) but may also
interact with Ala-
128(H6), which is involved in the
1-
1
contacts, and with Gly-
136(H14) located in the central cavity (12).
The known natural substitutions described at these two latter sites are
responsible for thermal or isopropyl alcohol instability (25). The
rupture of the
7(A4)-
132(H10) salt bridge near the
1-
1
interface and of
132(H10)-
128(H6) that directly participates in
the
1-
1 stabilization may induce a heat instability of the
mutated tetramers (Fig. 2). Among the three natural
132 mutants,
only Hb Cook (Lys-
132
Thr) has been shown to be slightly unstable (25).
Combinations of Hb S with another or
chain variant are
responsible for a variety of clinical patterns (26, 27). Information on
the location of intermolecular contacts in the polymer has been
obtained by studying the in vitro and in vivo
interactions of Hb S and natural Hb mutants (4, 5, 27). Hb K-Woolwich Lys-
132(H10)
Gln behaves like deoxy-Hb A when interacting with deoxy-Hb S, demonstrating that the
132 residue is not directly involved in the interactions stabilizing the deoxy-Hb S polymer (5,
28). Our results show that when associated with the sickle mutation on
the same
chain, neither the Lys-
132
Ala nor the Asn
substitution increases the hydrophobic interaction between donor and
acceptor sites. They both lead to a decrease in the maximum change in
absorption at 700 nm (Fig. 3) comparable to what was observed for the
rHb
E6V/E7A without significant modification of the solubility of
the rHbs (Fig. 6). The question arises whether these data account for
an inhibition of the polymerization process and/or for different
geometry or size of the polymers. It should be pointed out that the
"apparent" inhibition of the polymerization process is more
important when Lys-
132 is replaced by Ala than when replaced by Asn,
the inhibition being intermediate with the rHb
E6V/E7A. The
132
site is sterically near the acceptor pocket involving Phe-
85 and
Leu-
88. One may speculate that substituting the
132 residue
modifies the acceptor pocket and the fitting between the donor and
acceptor sites. In the rHb
E6V/K132A, two phenomena are susceptible
to interfere with the polymerization process. The absence of the salt
bridge as in the rHb
E6V/E7A would render the A helix softer and
would modify the acceptor pocket. As a result Val-
6 would not fit
well in the acceptor pocket, and the formation of the polymers would be
delayed. When Lys-
132 is replaced by Asn the consequences seem to be
less important. Molecular graphic modeling studies indicate that the
position of the Asn-
132 residue makes it possible to be
hydrogen-bonded to Glu-
7 (Fig. 7), thus maintaining
better donor-acceptor site contacts. Alternatively, although Val-
6
is essential to the interaction with the hydrophobic acceptor pocket,
other critical residues are involved in the stabilization of the
nuclei. Modifying the conformation of the A helix may prevent these
secondary contacts, resulting in an unstable nucleus. Note that the
value of Csat is similar to that found for Hb S
as also observed with rHb
E6V/E7A. The differences observed in the
aggregation rates for the
132 mutated rHbs (Fig. 5) relative to Hb S
and to the rHb
E6V/E7A may not only reflect quantitative but also
qualitative differences in the polymer formation.
Increasing the delay time (as observed for rHbs E6V/K132A and
E6V/K132N) would be of major interest to a therapeutic approach to
prevent Hb S polymerization in the microcirculation vessels. These
studies may help to determine critical target sites for antisickling
agents while preserving normal function and stability of the molecule,
which remains a challenge.
We thank M. Marden for much valuable discussion and helpful comments on the manuscript, L. Mouawad for help in the initiation to molecular graphic modelization studies, G. Caron and E. Domingues for skillful technical assistance, and T. Gorski for administrative care.