From the Department of Chemistry, Loyola University of Chicago, Chicago, Illinois 60626
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ABSTRACT |
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Spectrin of the erythrocyte membrane skeleton is
composed of Spectrin is the major component of the erythrocyte membrane
skeleton and is thought to be largely responsible for the red blood
cell's unique flexibility and deformability (1). Spectrin is composed
of two subunits, Amino acid (12) and cDNA (13) sequence analyses show that both The initial, antiparallel, side-by-side association of During the association of two dimers to form tetramers, it has been
suggested that the major interaction involves the bundling of the
N However, there are certain objections to assigning a preeminent role to
this helical bundling association in the tetramerization reactions. One
objection can be made on theoretical grounds, based on a proposed
structure of spectrin dimer. It has been suggested that in the normal
- and
-spectrin, which associate to form heterodimers
and tetramers. It has been suggested that a fractional domain (helix C)
in the amino-terminal region of
-spectrin (N
region) bundles with
another fractional domain in the carboxyl-terminal region of
-spectrin (C
region) to yield a triple
-helical bundle and
that this helical bundling is largely responsible for tetramer
formation. However, there are certain objections to assigning a
preeminent role to this helical bundling in the tetramerization
reactions. We prepared several recombinant peptides of
-spectrin
fragments spanning only the N
region (lacking the dimer nucleation
site) and quantitatively studied their interaction with
-spectrin.
We found that a majority of the interactions were localized, as
expected, in the N
-helix C region but that there was also some
contribution from the nonhomologous region. More importantly, the
temperature and ionic strength dependence of this interaction in our
model peptides was different from that in intact spectrin. We suggest
that, although the regions involving the putative helical bundling in
- and
-spectrin undoubtedly play a significant role in
tetramerization, regions distal to the N
-helix C region in spectrin
are also involved in tetramer formation. Structural flexibility and
lateral interactions may play a role in spectrin tetramerization.
INTRODUCTION
Top
Abstract
Introduction
References
-spectrin (280 kDa) and
-spectrin (246 kDa),
which associate to form
heterodimers (2, 3), which then
associate to form the biologically relevant (
)2
tetramers and higher order oligomers (4-6). The tetramer exists as a
flexible rodlike molecule that is anchored to the erythrocyte membrane by interactions with certain membrane proteins, most importantly band 3 and band 4.1 (7). This spectrin network imparts mechanical stability to
erythrocytes. Erythrocyte membranes in patients suffering from
hereditary spherocytosis and hereditary elliptocytosis exhibit unusually high mechanical fragility. A large subset of these diseases are known to be the result of defects in spectrin (8-11).
-
and
-spectrin are largely composed of multiple homologous motifs of
about 106 amino acid residues. These sequence motifs are suggested to
fold into triple
-helical bundles (structural domains) with the
first and third helices parallel and the intervening second helix
antiparallel (14). This proposed structure is supported by recent x-ray
(15) and NMR (16) studies of various recombinant spectrin fragments, as
well as by spectroscopic studies of intact spectrin (17-19). The three
helices show a pronounced amphipathic character, and the resulting
triple-
-helical bundle structure is thought to be stabilized by the
sequestration of the hydrophobic faces of each helix to the interior of
the bundle.
- and
-spectrin to form
dimers occurs at the amino terminus of
-spectrin and the carboxyl terminus of
-spectrin, with high affinity (Kd ~ 10 nM) (20). This site
is referred to as the dimer nucleation site. The rest of the
molecule is then thought to associate in a zipper-like fashion (21).
Two zipped dimers associate at the tetramerization site, the opposite
end of the molecule from the dimer nucleation site, resulting in a tetramer molecule (22). At this end of the molecule, both the amino-terminal region of
-spectrin (N
region)1 and the
carboxyl-terminal region of
-spectrin (C
region) have been shown
to encompass fractional 106-amino acid sequence motifs (23, 24). The
N
region contains a nonhomologous sequence, followed by a sequence
homologous to that of the third
-helix (helix C) in the motif (15).
According to our phasing studies with the first domain starting at
residue 52 (25), the fractional domain is coded for by amino acid
residues ~22-51 of the
-spectrin sequence. We will refer to this
fractional motif region as N
-helix C. It should be noted that there
is no direct experimental evidence that this fractional domain is or is
not entirely
-helical. This designation is made solely on the basis
of primary sequence homology (12). Similarly, the C
region contains
a nonhomologous segment and a longer fractional motif homologous to the
sequence of the first two
-helices in a complete motif.
-helix C with the two helices of the C
region to yield a triple
-helical bundle (23, 24). This helical bundling hypothesis is
bolstered by the fact that the majority of point mutations of spectrin
resulting in hereditary elliptocytosis occur in either the N
-helix C
region or the C
region with the two helices (10, 11, 26-29). In
most cases, it has been found that these spectrin mutants render
unstable tetramers.
dimer, the longer
-spectrin loops back on itself to form a
hairpin type structure to allow the helical bundling to occur (30). A
more elaborate view of the dimer-tetramer reaction can thus be
proposed, as shown in Fig. 1. In this
scheme, two N
/C
helical bundles are formed during
tetramerization, but two such bundles need to be broken first in each
of the hairpin dimers. Thus, the overall process is energetically
neutral with respect to helical bundling. Other interactions elsewhere
in the molecule must thus be responsible for driving the
tetramerization reaction. This is supported by the fact that, although
a majority of spectrin point mutations in hereditary anemias that cause
impaired tetramer formation are located in the N
-helix C region,
other such mutations are located distal to this region, at residues 260, 469, and 748, for example (7, 31).
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Fig. 1.
Model of the dimer association to form
tetramer. In this scheme, -spectrin is shown shaded,
while
-spectrin is shown unshaded. The individual
structural domains are delineated as rectangles (sometimes
distorted). For clarity, only the tetramer association region is shown;
in reality spectrin contains more such motifs and is much longer. It is
thought that isolated
dimers form hairpin loops (A),
which are stabilized by an
complex formation involving helical
bundling of the N
-helix C and the helices in the C
region. These
helical bundles need to be broken to form transient intermediates with
"sticky ends" (B) before they can reassociate to form
the (
)2 tetramer (C). Contributions to the
energetics of the process include not only the
and
association
(helical bundling) energy,
Gbundle, but also
possibly the energy needed to bend the
-spectrin extension back on
itself,
Ghairpin, as well as lateral
interactions between the adjacent
-spectrin regions of the tetramer,
G
-lateral. The overall energetics
(
Gtetramer:dimer) for this model is thus
independent of
Gbundle, with
Gtetramer:dimer = 2
Ghairpin
2
Gbundle + 2
Gbundle +
G
-lateral = 2
Ghairpin +
G
-lateral. It is possible that the
transition from B to C goes through another
intermediate form in which one of the helical bundles is formed while
the other is still open. However, this seems unlikely, since entropic
considerations would suggest that the second such interaction would be
more favorable than the first interaction between the two as yet
unlinked
dimers. In any case, these concerns in no way affect
the energetics of the system discussed above.
In order to examine this conundrum, we produced several well folded
recombinant -spectrin peptides spanning only the N
region (lacking the dimer nucleation site) and studied their interaction with
-spectrin quantitatively. These systems eliminated the nucleation interaction and the hairpin structure in
-spectrin to allow us to
focus on only the helical bundling interaction. We found that a
majority of the interactions in our
-spectrin peptides with
-spectrin were attributed to helical bundling in the N
-helix C
region. More importantly, the temperature and ionic strength dependence
of our
-peptides binding to
-spectrin was different from that in
intact spectrin. We suggest that, although the regions involving the
putative helical bundling in
- and
-spectrin undoubtedly play a
significant role in tetramerization, regions distal to the N
-helix C
region in spectrin are also involved in tetramer formation. Structural
flexibility and lateral interactions in these regions may play a role
in spectrin tetramerization.
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EXPERIMENTAL PROCEDURES |
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Preparation and Analysis of -Spectrin and of
-Spectrin
Peptides--
Spectrin dimer was obtained by low ionic strength
extraction of erythrocyte ghosts at 37 °C, followed by molecular
exclusion chromatography, as described previously (32).
-spectrin
was obtained from spectrin dimer as before (21), except that
-spectrin was purified on the FPLC system using MonoQ column
(Amersham Pharmacia Biotech) and NaCl gradient.
With the proper phasing for domain structure at residue 52 (25), we
constructed a series of recombinant -peptides, consisting of the
first three complete triple
-helical structural domains (residues
52-368), as well as (a) the entire leading nonhomologous region (residues 1-20) and the N
-helix C region (residues 21-51) to give peptide Sp
-(1-368), (b) half of the
nonhomologous region and the entire N
-helix C region
(Sp
-(12-368)), (c) only the N
-helix C region
(Sp
-(22-368)), (d) half of the N
-helix C region (Sp
-(37-368)), and (e) no N
-helix C region
(Sp
-(52-368)). In addition, we also prepared similar peptides with
only one (Sp
-(1-156)) or two (Sp
-(1-262)) complete structural domains.
The cDNA encoding the amino terminus of -spectrin (13) was used
as the template for polymerase chain reaction amplification of the gene
fragments. Pfu polymerase was used under standard conditions
to conduct both the polymerase chain reactions (33) and the mutagenesis
reactions (34). Fidelity of the polymerase-mediated reactions was
subsequently confirmed by direct DNA sequencing. Genes encoding for the
peptides Sp
-(1-368), Sp
-(1-262), Sp
-(1-156), and
Sp
-(52-368) were prepared as described previously (25, 32). The
genes encoding for the peptides Sp
-(12-368), Sp
-(22-368), and
Sp
-(37-368) were derived from the gene for Sp
-(1-368).
These peptides were expressed as glutathione S-transferase fusion proteins in Escherichia coli and released by thrombin cleavage with soluble thrombin (32). Purification procedures were as previously discussed (25, 32), except that the buffers for the FPLC MonoQ purification were changed to 25 mM bis-Tris at pH 6.0 and the NaCl concentration was changed to 500 mM. Peptide identity was checked by SDS-polyacrylamide gel electrophoresis and amino-terminal sequencing (Biocore Facility, University of Notre Dame, South Bend, IN). The molecular masses of these peptides were determined by mass spectrometry using electrospray ionization techniques (Washington University, St. Louis, MO). Protein concentrations were determined with absorbance values at 280 nm, using extinction coefficients determined from the primary sequence (32). In order to determine whether the peptides formed aggregates in buffer solution (5 mM phosphate buffer with 150 mM NaCl at pH 7.4), the solution molecular masses of the peptides at 1-2 mg/ml were determined at 4 °C with an absolute molecular mass determination instrument based on multiangle (15 and 90°) light scattering (PD2000; Precision Detectors, Inc., Amherst, MA) as described previously (32).
125I--Spectrin Peptides--
Interaction of
-peptides with the
-spectrin was examined by competition
experiments using unlabeled peptide to displace 125I-labeled peptide bound to
-spectrin. Iodination of
-peptides (100 µg) was performed in 100 mM phosphate
at pH 6.5, with IODO-BEAD (three beads; Pierce) and 2 mCi of
Na125I (ICN Pharmaceuticals, Costa Mesa, CA), followed by
dialysis, three times, against 10 mM Tris buffer at pH 7.4 with 20 mM NaCl and 130 mM KCl (isotonic buffer).
Binding Specificity--
A commonly used solid phase assay was
applied to study the affinities between various -peptides with
-spectrin. The isotonic buffer was used as the standard buffer and
the temperature of the reaction was 4 °C, unless stated otherwise.
In order to validate the binding specificity, we performed initial
studies with one of our larger N-terminal peptide fragments,
Sp
-(1-446) (25), and examined it for binding to spectrin dimer and
-spectrin. Wells of a microtiter plate (Immulon-4 from Dynatech,
Chantilly, VA) were coated with spectrin dimer or
-spectrin (~25
µg/ml, 16 h), and unreacted sites were blocked with either
bovine serum albumin (1 mg/ml) or nonfat powdered milk (9% w/v) for 4 h and then washed with the isotonic buffer. A constant amount of
125I-Sp
-(1-446) (~0.03 µM; ~50,000
cpm) was then added to these wells. Unlabeled Sp
-(1-446) (10 µM) was added to some wells to compete with the
125I-Sp
-(1-446), followed by washing with the isotonic
buffer containing 6% milk. The amount of
125I-Sp
-(1-446) remaining in each well was measured
with a
-counter (ICN Micromedic Systems).
To ensure sample equilibrium and to optimize the 125I
signal, the on and off time constants for this assay (on
and
off) of an
-peptide (Sp
-(1-368)) were also
checked. A constant amount of 125I-Sp
-(1-368) was added
to all wells, and the incubation period was varied from 0.5 to 50 h. The
on was found to be about 10 h
1. We
used this value to guide us to determine incubation time to achieve
equilibrium. Consequently, a standard incubation time of 16 h was
used for the assays.
The off rate was also examined to ensure that the equilibrium was not
disturbed while washing the microtiter wells. After incubation and
washing, buffer was added to the wells containing only the
125I--peptide and
-spectrin. The mixtures were
allowed to remain in the wells for different times, ranging from 1 min
to 6.5 h. We found that the dissociation was biphasic with ~20%
coming off with a
off ~10 min. The remaining
-peptide (~80%) came off much more slowly with a
off > 6 h. Therefore, there was not a significant disturbance of equilibrium during washing, which was accomplished in
<1 min.
Binding Affinity Assay--
-Spectrin (~25 µg/ml; ~0.1
µM) was added to wells of a microtiter plate for 16 h. Wells were washed, and unreacted sites were blocked with proteins in
nonfat powdered milk. Again, the isotonic buffer was used as the
standard buffer, and the temperature of the reaction was 4 °C,
unless stated otherwise. A constant amount of
125I-
-peptide (typically about 100,000 cpm,
corresponding to about 0.05 µM), was added to the wells
for interactions with the immobilized
-spectrin. Concurrently,
unlabeled
-peptide at various concentrations (0-20
µM) was added in isotonic buffer containing 6% milk, and the mixture remained in the well for 16 h. The unlabeled peptide competed with the 125I-labeled peptide for
-spectrin
binding sites. The solution was then removed, and the wells were washed
three times (in <1 min total time) with the buffer to remove nonbound
125I-labeled peptide. The amount of
125I-labeled peptide remaining in the wells
(cpmbound) was measured and found to decrease with
increasing concentrations of unlabeled peptides ([
]). A
displacement curve was obtained, and the values of IC50
(concentration of unlabeled peptide needed to inhibit/displace 50% of
the labeled peptide from binding the
-spectrin) were determined according to the equation (35, 36),
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(Eq. 1) |
For homologous competition experiments, the 125I-labeled
and unlabeled peptides were of the same type (e.g.
125I-Sp-(1-368) and unlabeled Sp
-(1-368)). For
heterologous competition experiments, different unlabeled peptides were
used to compete with the binding of 125I-Sp
-(1-368) to
-spectrin.
The IC50 in Equation 1 is similar to the more common solution dissociation constant (Kd) parameter used to characterize binding affinity in solution. Strictly speaking, affinities measured in this assay with one component of the binding reaction immobilized (IC50) are different from the true solution dissociation constants (Kd) (37). However, these types of affinity studies of soluble species toward solid phase species on microtiter plates (38, 39) or cell surface (40-42) have been widely reported and are considered a useful measure of binding strength. To recognize the different approaches between IC50 and Kd measurements, we used the term IC50 for our values, which were very similar to solution phase measurements of Speicher and co-workers (23, 43), when conditions of temperature and ionic strength were matched.
This assay can be used to conveniently screen -peptides with
different binding affinities toward the
-spectrin under the same
experimental conditions, since large numbers of assays can be set up
and analyzed at the same time. In the earlier chromatography-based assays (30), after the samples are incubated for long periods of time
to achieve equilibrium, the bound and the free ligand in the system are
separated by a column, which takes 30-50 min. In contrast, in this
assay, the separation time (washing time) is <1 min.
Ionic Strength and Temperature Dependence--
The binding of
Sp-(1-368) toward
-spectrin-coated plates was examined as a
function of ionic strength and temperature. The assays were conducted
at 4 °C in 10 mM Tris at pH 7.4 buffers with varying
amounts of added NaCl, ranging from 0 to 1 M, to give
different ionic strengths. The ionic strengths of these buffers were
calculated, including a ~7 mM contribution from the 10 mM Tris buffer, resulting in a range of 7-1007
mM. The milk solution used as a protein blocking solution
was also dialyzed against the appropriate buffer.
In the temperature studies, the samples in isotonic buffer were
incubated in the microtiter plates and washed at various temperatures between 0 and 30 °C.
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RESULTS |
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Protein Analysis--
The identity of -spectrin was confirmed
by SDS-polyacrylamide gel electrophoresis. The gel electrophoresis
molecular mass was ~250 kDa. Gel scan analysis of band intensities
indicated that the sample was at least 95% pure.
Gel electrophoresis molecular masses of the -peptides were ~18 kDa
for Sp
-(1-156), 31 kDa for Sp
-(1-262), 43 kDa for
Sp
-(1-368), 41 kDa for Sp
-(12-368), 40 kDa for Sp
-(22-368),
39 kDa for Sp
-(37-368), and 37 kDa for Sp
-(52-368). All seven
peptides were also analyzed by electrospray mass spectrometry and Edman
degradation of the amino terminus. Electrospray ionization mass
spectrometry of Sp
-(1-156), Sp
-(1-262), Sp
-(1-368),
Sp
-(12-368), Sp
-(22-368), Sp
-(37-368), and Sp
-(52-368)
demonstrated that the peptide masses were 18.67, 30.90, 42.93, 41.64, 40.67, 38.70, and 36.92 kDa, respectively, and were within 0.1% of the
theoretical molecular masses (18.67, 30.89, 42.94, 41.62, 40.65, 38.69, and 36.92 kDa, respectively). Amino-terminal sequencing (first 10 amino
acid residues) of the peptides confirmed that each peptide began with
residues GS, which remained from the thrombin cleavage site, and was
followed by the correct spectrin residues. The ratios of the solution
molecular mass, obtained from light scattering data, to theoretical
molecular mass for Sp
-(1-156), Sp
-(1-262), Sp
-(1-368),
Sp
-(12-368), Sp
-(22-368), Sp
-(37-368), and Sp
-(52-368)
in PBS were 0.97, 1.10, 0.97, 1.00, 1.00, 1.20, and 0.97, respectively.
These ratios show that most of the peptides in PBS at concentrations of
about 1 mg/ml existed as monomer. The Sp
-(37-368) peptide appeared to exhibit slight aggregation, since the ratio was about 1.2. The
-peptides were found by SDS-polyacrylamide gel electrophoresis to be
at least 90% pure.
The -helical contents from CD analysis of Sp
-(1-156),
Sp
-(1-262), Sp
-(1-368), Sp
-(12-368), Sp
-(22-368),
Sp
-(37-368), and Sp
-(52-368) were 57, 73, 76, 74, 75, 69, and
76%, respectively, based on a value of 36,000 for 100%
-helicity
(32). These helicity values indicated that the peptides were all well folded.
Binding Specificity--
As shown in Fig.
2, in isotonic buffer at 4 °C, upon
the addition of unlabeled Sp-(1-446), the amount of
125I-Sp
-(1-446) bound to spectrin dimer wells (457 ± 148 cpm for wells with Sp
-(1-446) and 502 ± 117 cpm for
wells without, n = 3) did not vary, whereas the amount
of 125I-Sp
-(1-446) bound to
-spectrin wells was
significantly reduced (1246 ± 373 cpm for wells without and
104 ± 25 cpm for wells with). These results showed that
Sp
-(1-446) bound specifically to
-spectrin but not to spectrin
dimers. Our spectrin dimers were prepared under conditions with the
dimers locked in the closed form (hairpin dimer) with helical bundling
of the fractional domains of
- and
-spectrin (21). Thus, the two
helices in
-spectrin in immobilized dimers were not available for
bundling with the helix in Sp
-(1-446). The 125I
activities found in the spectrin dimer wells, with or without unlabeled
Sp
-(1-446), probably reflected the nonspecific binding of
125I-Sp
-(1-446) to well surface that was not covered
with either spectrin dimer or blocking proteins (bovine serum albumin
or milk proteins). It appeared that spectrin dimer and
-spectrin
coated the wells of the titer plate differently, and thus different
amounts of residual nonspecific binding of
125I-Sp
-(1-446) to wells (502 ± 117 cpm for
spectrin dimer and 104 ± 25 cpm for
-spectrin as indicated
above) was observed. We also coated the plate with bovine serum albumin
and observed no significant difference in wells without (1689 ± 318 cpm) and in wells with (1558 ± 501 cpm) Sp
-(1-446),
further demonstrating binding specificity toward
-spectrin and not
other proteins.
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Binding Affinity--
For homologous displacement assays, Fig.
3A shows that all peptides
except Sp-(37-368) and Sp
-(52-368) exhibited displacement of
the labeled species by the unlabeled species, with IC50
values <1 µM in isotonic buffer at 4 °C. The average
IC50 values for these peptides are given in Table
I. All of the peptides beginning with the
first residue in the
-spectrin sequence, i.e.
Sp
-(1-156), Sp
-(1-262), and Sp
-(1-368), displayed
IC50 values of about 0.3 µM, with values for
Sp
-(1-368) being the lowest (0.24 ± 0.05 µM)
and Sp
-(1-156) being the highest (0.37 ± 0.15 µM). Since the experimental uncertainty was larger than
the difference, we could not be certain that the binding affinity of
Sp
-(1-368) toward
-spectrin was larger than that of
Sp
-(1-156).
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The peptides with the first 11 or 21 residues removed, Sp-(12-368)
and Sp
-(22-368), exhibited a slightly lower affinity toward
-spectrin, with IC50 values of 0.62 and 0.59 µM, respectively. For peptides with the first 36 or 51 residues removed, Sp
-(37-368) and Sp
-(52-368), no binding of
125I-peptides to
-spectrin was observed.
Heterologous displacement assays were also performed to ensure that the
different unlabeled -peptides displaced the
125I-Sp
-(1-368) peptide. Unlabeled Sp
-(37-368) and
Sp
-(52-368) did not displace 125I-Sp
-(1-368) at
concentrations up to 20 µM (Fig. 3B). These
results corroborated the nonbinding behavior in homologous displacement experiments for these two peptides. For the remaining peptides, the
IC50 values obtained from heterologous displacement curves were similar to those obtained from homologous displacement curves (Table I).
Ionic Strength and Temperature Dependence--
The binding of
Sp-(1-368) to
-spectrin showed no marked ionic strength
dependence at 4 °C (Fig. 4). The
measured IC50 values for the samples with ionic strengths
of 7 mM to 1 M were about 0.2-0.3
µM. However, the binding of the
-peptide to
-spectrin showed a strong temperature dependence in isotonic buffer
(Fig. 5). With increasing temperature,
the interaction weakened, from a value of ~0.2 µM at
4 °C to ~10 µM at 30 °C, a 50-fold decrease. The
interaction exhibited a nonlinear temperature dependence. The data,
log(IC50) versus T, was approximated well by a
quadratic relation (
log(IC50) = 6.490 + 0.019T
0.002T2, with T in °C). The measured pH in our Tris
buffer was 7.42 at 20 °C, 7.26 at 25 °C, and 6.99 at 35 °C.
For comparison of our data with those of the published work, in which
the Tris buffer at pH 7.4 was used for different temperature studies
(30), we also did not adjust the pH as we increased the
temperature.
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DISCUSSION |
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The association of - and
-spectrin at the tetramerization
site has been studied with intact spectrin (22, 23, 44, 45) as well as
with spectrin fragments (21, 23, 30, 43). It has been shown that a
proteolytically produced fragment,
I (21), and
-recombinant
peptides consisting of residues 1-639 (recombinant
I) or 1-158
bind
-spectrin, whereas peptides consisting of residues 31-639 or
28-158 do not bind
-spectrin (43). These results suggest that some
or all of the first 30 amino acid residues in
-spectrin are directly
involved in the tetramerization site interaction (43), and support the
hypothesis that the association involves helical bundling of fractional
domains in
- and
-spectrin (23, 24). However,
-peptides
containing just the fractional domain (residues 7-53) do not bind
-spectrin and do not appear to have appreciable native structure
(43).
We have recently demonstrated cooperative interactions between adjacent
structural domains in -spectrin peptides (32). This finding raises
the possibility that domains distal to the fractional domain
(N
-helix C) may influence the conformation of the N
-helix C
region and thus indirectly affect helical bundling and also
tetramerization. Quantitative binding parameters, such as
Ka, Kd, or IC50
values, of
-peptides with different sequences and/or conformations
interacting with
-spectrin are needed for comparison in order to
delineate various contributions to spectrin tetramerization. We applied
a commonly used solid phase assay to obtain quantitative
IC50 values of several
-peptides. These values were in
the micromolar range and agreed well with Kd values
(at 30 °C) of
-peptide consisting of residues 1-158 (43) and of
proteolytically digested
I fragment (21).
Our assay results at 4 °C in isotonic buffer showed that the binding
of Sp-(1-156) (peptide with one full domain) or of Sp
-(1-262) (peptide with two full domains), to
-spectrin was very similar to
that of Sp
-(1-368) (peptide with three full domains), with IC50 values of 0.2-0.3 µM. Thus, within
detection sensitivity, the second and third domains in
-spectrin did
not appear to affect the helical bundling at the N
/C
region. The
removal of either half (Sp
-(12-368)) or the entire
(Sp
-(22-368)) nonhomologous leader region reduced the affinity by
about 2.5 times when compared with Sp
-(1-368), with the
IC50 value increasing from 0.24 to 0.60 µM.
This leader segment thus appeared to play some role in binding
-spectrin. The majority of the interaction, however, appeared to be
mediated by the residues in the N
-helix C region (residues 21-51).
Elimination of half of this region (Sp
-(37-368)) substantially
reduced the binding affinity, with IC50 >20
µM. Thus, the dominant binding region in
-spectrin
started somewhere between residues 22 and 37. This is consistent with
previous results showing that the peptides starting at residue 28 or 31 did not bind to
-spectrin (43).
Speicher et al. (23) have proposed a "closed" form for
the dimer, with a hairpin bend in the -spectrin, the longer piece of
the two (
and
) subunits, when
- and
-spectrin associate to
form dimer. Since this form of spectrin has its "valency"
satisfied, it is unable to interact with another dimer to form
tetramers. Only the "open" form of the dimer, in which the hairpin
bend in
-spectrin is lost, is involved in tetramerization. This
model suggests a temperature barrier for dimer-tetramer interconversion caused by the conformational constraint of the dimer. In our systems, we studied only the interactions between N
and C
regions in spectrin (measuring only the helical bundling) and not the conversion of closed and open conformations discussed above. If helical bundling is the major interaction between
- and
-spectrin in the spectrin tetramer, our model system should mimic the binding properties of
spectrin dimers to form tetramers quite well. Otherwise, our system
would only mimic a part of the tetramerization interactions in intact spectrin.
In the model presented in Fig. 1, the overall reaction is dependent not
only on the N/C
interactions (
Gbundle)
but also on other interactions between the two
-chains in the region
distal to the immediate N
and C
regions
(
G
-lateral) as well as a term
related to the flexibility of the
-chain
(
Ghairpin). This scheme predicts that
the overall reaction is expected to be energetically neutral with
respect to the
Gbundle term, since two such
N
/C
interactions (helical bundling) are formed in the tetramer,
but two are also broken in each of the normally closed (hairpin)
dimers.
G
-lateral contributions have been
hinted at by the identification of certain known spectrin defects
located well outside the N
helix C region that result in defective
erythrocyte membranes (7, 31). Thus, we propose that, while the helical bundling is involved in the dimer association to form tetramers, other
interactions (
G
-lateral and
Ghairpin) are also important. This suggestion
is supported by the results obtained from our temperature and ionic
strength experiments. IC50 values for the association of
our peptides with
-spectrin remained constant (~0.2
µM) over the range 7 mM to 1 M.
The helical bundling in the N
/C
region has been hypothesized to
be largely due to hydrophobic effects, since the interior of these
three
-helix bundles can bee seen to be populated by a hydrophobic
amino acid sequence (15). Hydrophobic effects are generally
strengthened (46, 47), whereas electrostatic interactions are weakened
by increases in ionic strength. The interactions observed in this study
appeared to be not affected by ionic strength, implying that a balance of hydrophobic and electrostatic forces may be at work. Such behavior has been seen in interactions of certain other synthetic peptides of
-helical coiled coils (44). However, the intact dimer to tetramer
equilibrium shows a different behavior; the association weakens with
increasing ionic strength (48, 49). The interaction in intact spectrin
is complicated by the fact that many species are present, including not
only dimers and tetramers but also higher order oligomers (as well as
some monomers under certain conditions). This makes the exact
association constants measured somewhat model-dependent.
However, in all cases, the association decreases with ionic strength.
Ultracentrifugation studies (49) have shown that the dimer to tetramer
association constant, K2,4, in the SEKIII model
(cooperative isodesmic model (50)) in which the dimer self-associates
to form a tetramer with an equilibrium constant
K2,4 (which is larger than that,
Kiso, which governs all successive additions of
the heterodimer) varies from ~1.2 × 106
M
1 (corresponding to a Kd
of ~0.8 µM) to ~6 × 104
M
1 (Kd of ~16
µM) as the ionic strength is varied from 90 mM to 1 M (see Fig. 5B in Ref. 49).
Our
-peptides did not display this behavior. The binding affinity in
our model peptides remains constant at IC50 ~ 0.2 µM over the ionic strength range of 7 mM to 1 M. These results suggested that, in intact spectrin, interactions other than helical bundling are involved in tetramer formation.
The temperature dependence of the interaction may provide a clue to the
role of the - and
-spectrin association in tetramerization. It is
known that the tetramer-dimer equilibrium is
temperature-dependent. Tetramers are stabilized by low
temperature (e.g. 4 °C), while dimer formation is
promoted at higher temperatures (e.g. 37 °C). At low
temperature, the affinity of our Sp
-(1-368) (IC50 = 0.24 µM at 4 °C) was very similar to that of
proteolytically digested
I fragment (consisting of residues 1-639)
(Kd ~ 0.22 µM at 0 °C) (30).
However, the temperature dependence was not so marked for this
I
fragment (Kd increasing to ~3.8 µM
at 30 °C) (30), whereas our peptide exhibited a strong temperature dependence (IC50 of Sp
-(1-368) was increased to 10 µM at 30 °C). At 37 °C, our extrapolated value for
IC50 was estimated to be 60 µM, implying that
at physiological temperatures helical bundling would be weak. A similar
reduction in affinity at physiological temperatures was also reported,
but in less quantitative form, using a related
-spectrin peptide in
a different assay system (43). Again, the properties of the dimer to
tetramer association of intact spectrin differed. For intact dimer to
tetramer association over this temperature range, ultracentrifugation
studies show that the association was weakened only slightly upon
increasing temperature, with Kd ~0.5
µM at 20 °C and ~1.7 µM at 35 °C
(44). At lower temperatures (e.g. 4 °C), kinetic trapping prevents the dimer-tetramer equilibrium from being achieved on experimental time scales, so reliable information about the
Kd of intact spectrin at these low temperature is
not available. However, the dimer association in intact spectrin is
much stronger (Kd ~1.7 µM at
35 °C) than the
-peptide/
-spectrin association (helical
bundling) that we (~10 µM at 30 °C or our
extrapolated value of ~60 µM at 37 °C) and others
have observed, suggesting additional interaction in the intact spectrin system.
What then is the role of this N/C
helical bundling interaction?
Is this simply an in vitro curiosity that has little
relevance for the in vivo behavior of spectrin? The
temperature behavior is consistent with the role of this association
being responsible for the observed kinetic barrier to
dissociation. This kinetic barrier is large and is responsible for the
well known trapping of the dimer form of spectrin at low temperatures
(30), although their association to tetramers is thermodynamically
favorable. Although the scheme in Fig. 1 shows that the overall
equilibrium in the 2
[dharrow] (
)2 reaction
should be independent of helical bundling
(
Gbundle), the reaction still must proceed
along a reaction path that requires this energy to be put into the
reaction. In fact, since two such interactions must be broken for a
complete rearrangement, the dissociation kinetics should be doubly
sensitive to this energetic term, perhaps even determining the kinetics of rearrangement of the spectrin molecules within the erythrocyte membrane skeleton. The in vivo relevance is thus apparent,
since this rearrangement behavior is likely to be linked to the
erythrocyte deformability.
In summary, we demonstrated that in -spectrin the region consisting
of residues 12-51 was involved in binding with
-spectrin, primarily
via helical bundling of the region consisting of residues 20-51.
However, the major significance of this work lies not in that assertion
but in the demonstration that the helical bundling interactions in
-peptide/
-spectrin, in particular the ionic strength and
temperature dependence, are dissimilar to those previously observed for
the dimer-dimer interaction in native spectrin. The temperature
dependence suggests that the function of this limited interaction
(helical bundling) is to provide a kinetic barrier in the overall dimer
to tetramer conversion and contributes to the previously demonstrated
temperature dependence of this association. This region may thus
control the rearrangement behavior of the spectrin molecules within the
membrane skeleton. We suggest that regions distal to the N
-helix C
region in spectrin are also involved in tetramer formation. Structural
flexibility and lateral interactions in these regions may also play a
role in spectrin tetramerization.
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ACKNOWLEDGEMENTS |
---|
The cDNA clone, 3, used to produce the
recombinant spectrin fragments was a gift of Dr. B. G. Forget
(Yale University School of Medicine, New Haven, CT). We thank Dr. D. Speicher of the Wistar Institute for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Science Foundation Grant NSF-MCB9407779 (to L. W. M. F.), a grant from American Heart Association Metropolitan Chicago (to N. M. and L. W. M. F.), a Department of Education GAANN Fellowship (to L. C.), and an American Heart Association Metropolitan Chicago Senior Fellowship (to N. M.).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.
To whom correspondence should be addressed: Dept. of Chemistry,
Loyola University of Chicago, 6525 N. Sheridan Rd., Chicago, IL 60626. Tel.: 773-508-3128; Fax: 773-508-3086; E-mail: lfung{at}luc.edu.
The abbreviations used are:
N region, N
amino-terminal region of
-spectrin; C
region, C
carboxyl-terminal region of
-spectrin; Sp
-(x-y), recombinant peptide with sequence homologous to that of human
erythrocyte
-spectrin, starting at residue x and ending
at residue y; FPLC, fast protein liquid chromatography; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
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REFERENCES |
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