(Received for publication, February 16, 1995; and in revised form, May 31, 1995)
From the
The spectrin-actin-binding domain of protein 4.1 is encoded by a 21-amino acid alternative exon and a 59-amino acid constitutive exon. To characterize the minimal domain active for interactions with spectrin and actin, we functionally characterized recombinant 4.1 peptides containing the 21-amino acid cassette plus varying portions of the 59-amino acid cassette (designated 21.10 to 21.59). Peptide 21.43 was shown fully functional in binary interactions with spectrin (by cosedimentation and coimmunoprecipitation experiments) and in ternary complex formation with spectrin and actin (by an in vitro gelation assay). Further truncation produced peptides incapable of binary interactions but fully competent for ternary complex formation (peptides 21.36 and 21.31), shorter peptides with reduced ternary complex activity and altered kinetics (21.26 and 0.59), and inactive peptides (21.20 and 21.10). Binding studies and circular dichroism experiments suggested that residues 37-43 of the constitutive domain were directly involved in spectrin binding. These data indicate that 4.1-spectrin binary interaction requires the 21amino acid alternative cassette plus the 43 N-terminal residues of the constitutive domain. Moreover, the existence of two possible ternary complex assembly pathways is suggested: one initiated by 4.1-spectrin interactions, and a second by 4.1-actin interactions. The latter may require a putative actin binding motif within the 26 N-terminal residues of the constitutive domain.
The erythrocyte membrane mechanical properties are determined by
a protein skeletal network underlying the lipid bilayer composed mainly
of spectrin, actin, protein 4.1, protein 4.9, and adducin(1) .
The ternary interaction between spectrin tetramer, actin protofilament,
and protein 4.1 forms a highly stable complex (K
10
M
)
which imparts mechanical integrity to the
membrane(2, 3, 4) . While the different
binary interactions between spectrin, actin, and protein 4.1 have been
extensively studied, little is known about the mechanism of the ternary
complex formation. Binary interaction between spectrin and actin is
characterized by a weak binding (K
5
10
M
)(3, 5) . While the
interaction between protein 4.1 and F-actin was initially found to be
weak(5) , more recent studies suggest a cooperative mechanism
for this interaction(6) . (
)In contrast, protein 4.1
binds strongly to spectrin (K
ranging
from
0.5 to 86
10
M
)(7, 8, 9, 10) .
Although both subunits of spectrin are required to form a ternary
complex with protein 4.1 and actin, only
-spectrin determines the
sensitivity of the spectrin-actin interaction with protein
4.1(11, 12) .
Alternative pre-mRNA splicing
generates a number of protein 4.1 isoforms in a tissue- and
development-specific manner(13, 14, 15) . A
10-kDa domain of protein 4.1 has been shown to be required for
spectrin-actin interactions(16) . This domain is encoded by an
alternatively spliced exon (encoding the N-terminal 21-amino acid (aa)) ()and a constitutive exon (encoding the C-terminal 59
aa)(17) . Expression of the 21-aa alternative exon occurs late
in erythroid development and is concomitant with major changes in red
cell membrane properties(18, 19) . The 21-aa cassette
is required for binding of protein 4.1 to spectrin, for stabilization
of spectrin-actin complex, and for imparting mechanical strength to the
membrane(20, 21) .
The aim of the present study is to determine if the 21-aa cassette by itself constitutes the minimal functional domain required for spectrin-actin interactions. To achieve this objective, we have expressed, characterized, and functionally assayed a nested set of truncated peptides designated 21.10 to 21.59, containing the 21-aa plus variable portions of the C terminus of the spectrin-actin-binding domain. Using both cosedimentation and immunoprecipitation methods, we have shown that binding of recombinant peptides to spectrin requires the 21-aa cassette plus at least 43 residues of the 59-aa domain; no interaction with spectrin was detected using shorter peptides. This minimum sequence of 64 aa was also required for the formation of a ternary complex manifested by gelation at physiologically relevant conditions. However, peptides further truncated from the N terminus (e.g. 0.59) or from the C terminus (21.26) could promote gelation of spectrin-actin mixtures at much higher concentrations. These truncated peptides may initiate ternary complex formation via an interaction with actin mediated by a motif within the first 26 residues of the 59-aa domain. The peptides 21.20 and 21.10 had no function whatsoever, suggesting that the alternative 21-aa cassette in itself is inactive. We have thus characterized the sequences in protein 4.1 involved in its interactions with spectrin and actin.
Figure 1:
Map of the
protein 4.1 recombinant peptides. A, schematic diagram of the
10-kDa domain of protein 4.1. Residues 406-472 of protein 4.1
were previously identified as the spectrin-actin-binding domain of
protein 4.1(26) . The alternatively spliced 21-aa cassette and
the constitutively expressed 59-aa cassette are identified.
Phylogenetically conserved sequences are in gray. The
indicated phosphorylation sites (at Tyr
and
Ser
) have been previously
described(41, 43) . B, the different
COOH-terminal truncations of the prototypical erythroid 4.1 10-kDa
domain isoform termed 21.59. The construct 19.59 containing a
non-erythroid alternatively spliced cassette and the construct 0.59
lacking the 21-aa cassette are also
represented.
Figure 2: Bacterially expressed spectrin actin-binding domain peptides of protein 4.1. 10 µg of reverse phase HPLC-purified thrombin-cleaved peptides were subjected to 20% SDS-PAGE and stained by Coomassie Blue.
Figure 3:
Cosedimentation of S-labeled
GST peptides with spectrin. The binary interaction of
S-labeled GST constructs at indicated concentrations with
spectrin (6 µM) was assayed by cosedimentation as
described under ``Materials and Methods.'' Sedimentation of
spectrin dimer is represented by a horizontal line in these
panels. A, sedimentation profile of
[
S]GST-(21.43, 21.26, 21.10) with spectrin; B, sedimentation profile of
[
S]GST-0.59; C, sedimentation profile
of [
S]GST-21.43 at 3 and 30 µM. The
control corresponded to the typical sedimentation profile of peptide
[
S]GST-21.43 (3 µM) without
spectrin.
To demonstrate the specificity of the binary interaction
between the fusion peptides and spectrin, the cosedimentation of
[S]GST-21.43 (3 µM) with spectrin
(6 µM) was assayed in the presence of excess of unlabeled
fusion proteins (Fig. 4). Only GST-21.43 inhibited sedimentation
of the [
S]GST-21.43 with spectrin; 25 µM of GST-21.43 induced a
75% inhibition. Neither GST-21.26 at
45 µM, nor the chemically synthesized 21-aa peptide at 55
µM induced any inhibition. These results suggest that the
interaction between [
S]GST-21.43 and spectrin
was specific. Both binding and inhibition data suggest that while the
21-aa cassette is necessary for binding to spectrin, it is not
sufficient. Additional 43 residues of the constitutive domain along
with the 21-aa cassette are required for spectrin binding in this
binary assay.
Figure 4:
Inhibition of cosedimentation of S-labeled GST constructs with spectrin. The spectrin (6
µM) was incubated with excess of thrombin-cleaved peptides
21.43, 21.26, and a synthetic 21-aa peptide, prior to perform
cosedimentation experiment in the presence of
[
S]GST-21.43 (3 µM), as described
under ``Materials and Methods.'' The control corresponded to
the amount of bound [
S]GST-21.43 in the absence
of inhibitor. The error bars indicate the variability of the
method determined during the course of three different
experiments.
To examine more labile interactions between peptides
and spectrin that might not be detected by the slow sucrose gradient
separation of bound and free species (12 h), we used a rapid
coimmunoprecipitation assay (separation 15 min) (Fig. 5). An
antibody directed against the GST moiety was used to immunoprecipitate
GST-4.1 fusion proteins, and the amount of cosedimenting
[
H]spectrin was quantified to estimate binding
affinity. Consistent with above results, only peptide GST-21.43 had a
moderate affinity for [
H]spectrin (K
0.5
10
M
). GST-21.36 and GST-21.31 bound
very weakly to [
H]spectrin while shorter peptides
and GST-0.59 did not bind to [
H]spectrin (Fig. 5A). Equivalent results were obtained using 15
µM of anti-GST antibody (data not shown).
Figure 5:
Coimmunoprecipitation of
[H]spectrin with recombinant 4.1 peptides. A, incubation of purified [
H]spectrin (2
µM) with various GST fusion proteins (1.6 µM)
was conducted as described under ``Materials and Methods.''
The notation gel refers to the results of a viscometric assay
run in parallel with the indicated peptides at 2.5 µM,
spectrin (1.8 µM), and actin (14 µM). In the
control reaction, the GST construct was replaced by the GST moiety
alone. B, inhibition assay of
[
H]spectrin immunoprecipitation. The
[
H]spectrin (2 µM) was preincubated
with excess of protein 4.1 and various thrombin-cleaved peptides prior
to mixing with GST-21.43 (1.6 µM). The inhibition
activities of, respectively, 21.43, 21.26, 0.59, and an antibody anti
21 at indicated concentrations were measured in two different
experiments. The control reaction was performed in the absence of
inhibitor.
To further
confirm the specific binding of 21.43 to spectrin, we performed
additional inhibition studies. The cosedimentation experiment was
conducted in the presence of excess of thrombin-cleaved peptides to
rule out the contribution of the GST moiety (Fig. 5B).
The cleaved peptide 21.43 induced a dose-dependent inhibition of
binding of GST-21.43 (1.6 µM) to
[H]spectrin (2 µM). The 21.43
induced, at 5 µM,
65% inhibition, and at 20
µM, an almost complete inhibition, similar to the effect
of full-length protein 4.1 at 11 µM. In contrast, the
cleaved peptides 21.26 and 0.59 did not inhibit binding of the
GST-21.43 to [
H]spectrin, up to concentrations of
48 µM and 250 µM, respectively. Finally, an
anti 21 antibody also strongly inhibited binding of the GST-21.43 to
[
H]spectrin (Fig. 5B). This
antibody inhibition could be due to either masking of a binding site in
the 21-aa cassette and/or steric interference with binding site(s) in
the 59-aa domain.
Taken together, these results imply that the 21-aa alternatively spliced cassette plus the 43 N-terminal aa of the constitutive cassette are required for a specific interaction of protein 4.1 with spectrin in solution.
Figure 6:
Induction of in vitro gelation of
spectrin-actin mixture by recombinant 4.1 peptides. A,
relative effect of various GST fusion proteins on the gelation of
spectrin (1.8 µM) and actin (14 µM).
Viscosity of the mixtures was measured as described under
``Materials and Methods.'' The gelation activity
(µM) was calculated for each fusion
peptide as the inverse of the minimum peptide concentration inducing
gelation of the spectrin-actin mixture. The control consisted of a
mixture of actin and spectrin alone. B, effect of cleaved
peptides on the kinetic of gelation. Mixture of spectrin (1.8
µM) and actin (14 µM) were incubated on ice
with cleaved 21.43, 21.26, and 0.59 at indicated concentration range.
For each concentration tested, the time necessary to form a gel was
measured by falling ball viscometry. The gelation velocity corresponds
to the inverse of the gelation time.
The ability of peptides shorter than 21.43 to induce ternary complex formation, while being unable to bind spectrin, suggested that different mechanisms may be operative in inducing gel formation by 21.43 versus these other peptides.
To explore whether
putative different ternary complex assembly pathways might exhibit
different kinetics, the effect of increasing amounts of cleaved
peptides on the gelation velocity of a spectrin-actin mixture was
assayed. Two types of responses were noted. Increasing amounts of
peptide 21.43 induced a logarithmic enhancement of the rate of gelation (Fig. 6B). In marked contrast, the rate of gelation
observed with peptide 0.59 was much slower, reaching a maximum value at
120 µM. Peptide 21.26 showed a response similar to
the 0.59, albeit leveling off at 90 µM (Fig. 6B). These kinetic data support the thesis
that different mechanisms of gel formation indeed exist.
To quantify
more precisely the potency of various GST-recombinant peptides to
incorporate spectrin into the ternary complex, the composition of the
gelation mixtures was quantified by a cosedimentation assay. A mixture
of 1.8 µM spectrin and 14 µM actin alone did
not exhibit increased viscosity (see Fig. 5A). Under
these conditions, 35% of the total spectrin cosedimented with
95% of the actin, indicating a weak binary interaction between
spectrin and actin. In contrast, in the presence of 4.1 peptides, a
correlation was observed between increase in viscosity and increased
incorporation of spectrin into the ternary complex. In the presence of
GST-21.43 at a concentration of
5 µM,
90% of the
spectrin was incorporated into the ternary complex. In contrast,
20 µM and
50 µM of GST-21.26 and
GST-0.59, respectively, were necessary to incorporate
50% of the
spectrin in a ternary complex. Peptides GST-21.20 and GST-21.10 did not
increase the incorporation of spectrin with actin up to concentrations
of
55 µM. In all these experiments, 95% of actin was
recovered in the pelleted fraction. These results further support the
idea that different mechanisms of ternary complex formation may be
operative depending on the affinities of the peptides for spectrin.
To
further characterize the secondary structure of the peptides, CD
measurements were performed on the HPLC-purified peptides 21.37 and
21.43, as well as on a gel filtration-purified mixture of these two
peptides. CD spectra indicated that both peptides exhibit similar
secondary structures (Fig. 7). These results imply that 1)
removal of 6 aa from 21.43 does not detectably alter its secondary
structure and 2) these residues are directly involved in spectrin
binding rather than maintenance of proper 4.1 conformation. Analysis of
the curves gave a maximal -helical content of <5% for both
21.37 and 21.43; a value significantly less than predicted from the
primary structure analysis. Indeed, according to the method of Garnier et al.(35) , predicted
-helical contents were 72
and 68% for 21.37 and 21.43, respectively. CD analysis of the mixture
of 21.37 and 21.43, purified by an alternative gel filtration
technique, also showed a low
-helical content suggesting that the
HPLC purification procedure did not alter the secondary structure of
the peptides (data not shown). To determine the intrinsic propensity of
the gel-filtration purified peptides to adopt a helical conformation,
the CD spectra were measured in the presence of 40% TFE and 1% SDS as
inducers of helicity (36, 37, 38, 39) and of 1% Tween 20
as a negative control. Secondary structure analysis gave
42%
-helix with TFE and
26% with SDS while Tween 20 did not alter
secondary structure (data not shown). The large increase in helicity
obtained with TFE suggests that both peptides have the propensity to
adopt a helical conformation.
Figure 7: Circular dichroism spectra of peptides 21.37 and 21.43. Circular dichroism spectra measured for thrombin-cleaved HPLC-purified peptides 21.43 and 21.37, at room temperature, in buffer B. Samples were prepared as described under ``Materials and Methods.''
We have investigated the structural requirements for protein 4.1 binary interactions with spectrin, as well as for ternary interactions with spectrin and actin. Analysis of different binding behavior observed among a nested set of truncated recombinant 4.1 peptides in several in vitro assays (sedimentation, immunoprecipitation, and viscometry) have enabled the development of a model for ternary complex assembly. Our data are consistent with a model in which a core actin binding motif is flanked by two sequence elements required for high affinity spectrin binding; details of this model and supporting evidence are discussed below.
Both binding and
inhibition studies showed that the peptide 21.43 specifically bound
spectrin with a moderately high affinity (K
0.2-0.5
10
M
). This affinity for spectrin is
similar to the 0.5
10
M
value previously reported using similar methods for both the
prototypical SAB construct 21.59 and the full-length protein
4.1(9, 20) . Deletion of additional N- or C-terminal
residues (0.59 and 21.36, respectively) led to loss of spectrin
binding. Thus, residues 407-427 (the 21-aa cassette) and
464-470 (the difference between 21.36 and 21.43) are identified
as two key motifs essential for a high affinity binding to spectrin.
Interestingly, the physiological importance of these motifs is also
supported by the observations that both are highly conserved
evolutionarily, and both include phosphorylation sites previously
reported to decrease 4.1-spectrin-actin interactions. Sequence
comparison of 4.1 from mammalian (human, mouse, dog), amphibian (frog),
and avian (chicken) sources has shown that residues 414-429 and
457-469 are highly conserved(40) . (
)Moreover,
phosphorylation of Tyr
within the 21-aa cassette has been
previously identified as disruptive to spectrin-actin-4.1
interactions(41) . In addition, phosphorylation of protein 4.1
on Ser
within the 59-aa domain also decreased the ternary
complex formation and the protein 4.1-spectrin
interaction(42, 43) . The dramatic decrease in
spectrin affinity as the 21.43 is shortened at its C terminus may thus
be related to the loss of highly conserved amino acids including
Ser
.
Requirements to form a ternary complex with
spectrin and actin were different than requirements for binary
interaction with spectrin. One class of peptides, represented by 21.36
and 21.31, exhibited minimal interaction with spectrin in binary
assays, but nevertheless enhanced spectrin cross-linking of actin
filaments with activities similar to that of 21.43 and full-length 4.1.
These peptides induced formation of a gel at physiological molar ratios
(dimeric spectrin/actin/4.1 peptide 1:7:1). We interpret these
results to indicate that the highly conserved residues 31-36 of
the constitutive domain, while not allowing strong binary interaction
with spectrin, nevertheless play a role in stabilizing spectrin binding
in the presence of actin. Further truncation produced a second class of
peptides (0.59 and 21.26) that was also inactive in spectrin binary
assays but exhibited a qualitatively and quantitatively different
ternary complex activity. These peptides were active only at
concentrations 10-50-fold higher than normal 4.1 and much higher
than the estimated ``cellular'' concentration (
200,000
molecules in a cell volume of
100 femtoliters, i.e.
3 µM). Although, we note that the red cell 4.1
can be concentrated at the membrane by additional interactions with
glycophorin C, p55 and band 3 (44, 45, 46, 47, 48) ;
therefore, the effective membrane skeletal concentration of 4.1 is
likely higher than 3 µM. In addition, this class of
peptides exhibited different kinetics of gel formation, implying a
different molecular basis for ternary complex formation. We speculate
that the central domain shared by peptides 0.59 and 21.26, i.e. the first 26 residues of the 59-aa domain (amino acids
428-453 of intact 4.1) may induce ternary complex formation by
functioning as an actin-binding site. Hence, a weak binary interaction
with F-actin involving this motif might initiate ternary complex
formation in vitro, even in the absence of high affinity
spectrin binding. The importance of this putative actin motif is
supported by 1) the fact that the shorter peptides 21.20 and 21.10,
lacking part of the putative actin binding motif, displayed no activity
in any of the assays, and 2) the recent observation that a protein 4.1
variant with a deletion of one residue from the Lys
doublet (corresponding to residues 20-21 of this actin
binding motif) is unable to interact with spectrin or spectrin-actin
mixtures(49) .
Consistent with this model, a cooperative
actin binding activity with protein 4.1 has been reported earlier using
cosedimentation assays (6) . Moreover, our
preliminary experiments with these peptides have also shown a binary,
cooperative association with F-actin (data not shown). Furthermore,
cosedimentation experiments with excess of actin and limiting amounts
of spectrin also provide evidence consistent with cooperative 4.1:actin
binding. Peptides 21.43, 21.26, and 0.59 sedimented in molar excess of
the available spectrin in this assay. All three peptides exhibited
cooperative binding and reached saturation at approximately the total
actin concentration, implying an additional cooperative interaction
with F-actin. (
)
The discrepancies between the calculated
and experimentally determined Stokes radii and also between the
predicted and measured secondary structures suggest that the peptides
21.43 and 21.37 do not require a compactly folded structure for
activity. While capable of initiating gelation in buffer B, both these
peptides exhibited an helicity substantially lower than predicted (this
report and (31) ). This difference may be attributed to the
fact that short polypeptides in solution can be disordered by polar
interactions between water and the peptide backbone(38) .
Previous studies with purified proteins, using in vitro binding assays, suggested a conformational change of one or
several components of the ternary complex(3) . A conformational
readjustment was recently posited for recombinant peptides
corresponding to the putative actin-binding site of Nebulin (50) . Mechanistically, the acidic F-actin molecule might
increase helicity of bound 4.1 peptide in similar fashion. Additional
experiments using known inducers of helical content trifluoroethanol
and SDS (37, 38, 51) indicate that peptides
21.43 and 21.37 have the propensity to adopt an -helical
conformation (data not shown). Further structural studies by
two-dimensional NMR will be necessary to establish whether the TFE- and
SDS-induced helicity we observed is of biological importance, and
whether conformational adjustments of protein 4.1 occur upon actin and
spectrin binding.
Based on the present studies as well as previous solution binding assays and visualization of protein-protein interactions by electron microscopy(3, 5, 52) , we propose the following model of ternary complex assembly. Although formation of a ternary complex can arise from simultaneous interaction among three proteins, it is more likely that the ternary complex assembly begins with a binary complex. Three pathways (noted I, II, and III) schematize the possible binary interactions which initiate assembly of the ternary complex, i.e. 4.1 peptide-spectrin (I), 4.1 peptide-actin (II), and spectrin-actin (III). Our results suggest the following scenarios for the assembly of the ternary complex involving different peptides. For protein 4.1 and peptides that can strongly bind to spectrin, e.g. 21.43 and 21.59, pathway (I) would be the dominant mechanism of ternary complex assembly. This pathway would represent the physiological mechanism corresponding to the rapid binding of peptide 21.43 to spectrin followed by the rapid cross-linking of F-actin. This pathway displayed no rate limitation in the kinetic study suggesting a rapid assembly of the ternary complex. In contrast, the smaller peptide 21.26, and also the nonerythroid peptides 0.59 and 19.59, do not have the strong affinity for spectrin necessary for initiating pathway (I) and would thus follow pathway (II). In this pathway, the peptides first bind cooperatively to F-actin, and this complex interacts slowly with spectrin. Indeed, the apparent saturation in gelation speed with increasing concentrations of 21.26 and 0.59, at fixed spectrin concentration, suggests a rate limitation most consistent with a slow spectrin cross-linking of peptide-actin complexes represented in pathway (II). For this reason, the peptides 21.31 and 21.36, while inducing ternary complex assembly with a potency similar to that of 21.43 and because of their low affinity for spectrin, would also follow pathway (II). In pathway (III), a weak interaction of spectrin with actin is followed by the binding of any functional peptide. The weak interaction of spectrin with actin is emphasized by the weak cosedimentation of spectrin with actin in the absence of peptide and also by the low viscosity of a spectrin-actin mixture.
Our data suggest the following domain map of
this 64 aa sequence: two spectrin-binding sites (the 21-aa cassette and
the residues 27-43 of the constitutive domain) flank an
actin binding site (first 26 residues of the constitutive domain).
These putative binding sites contain charged residues, consistent with
localization of the SAB to the surface of protein 4.1. Furthermore, we
have shown that the 21-aa cassette is not a functionally autonomous
structure, possessing the complete spectrin-actin binding activity of
the erythroid 4.1, but does appear to be a strong modulator of this
function.