(Received for publication, July 28, 1995; and in revised form, December 1, 1995)
From the
Human erythroid spectrin dimer assembly is initiated by the
association of a specific region near the N-terminal of -spectrin
with a complementary region near the C-terminal of
-spectrin
(Speicher, D. W., Weglarz, L., and DeSilva, T. M.(1992) J. Biol.
Chem. 267, 14775-14782). Both spectrin subunits consist
primarily of tandem, 106-residue long, homologous, triple-helical
motifs. In this study, the minimal region of
-spectrin required
for association with
-spectrin was determined using recombinant
peptides. The start site (phasing) for construction of dimerization
competent
-spectrin peptides was particularly critical. The
beginning of the first homologous motif for both
-spectrin and the
related dimerization site of
-actinin is approximately 8 residues
earlier than most spectrin motifs. A four-motif
-spectrin peptide
(
1-4
) with this earlier starting point
bound to full-length
-spectrin with a K
of about 10 nM, while deletion of these first 8
residues reduced binding nearly 10-fold. N- and C-terminal truncations
of one or more motifs from
1-4
showed that
the first motif was essential for dimerization since its deletion
abolished binding, but
1
alone could not
associate with
-monomers. The first two motifs
(
1-2
) represented the minimum lateral dimer
assembly site with a K
of about 230
nM for interaction with full-length
-spectrin or an
-spectrin nucleation site recombinant peptide,
18-21.
Each additional motif increased the dimerization affinity by
approximately 5-fold. In addition to this strong inter-subunit dimer
association, interactions between the helices of a single
triple-helical motif are frequently strong enough to maintain a
noncovalent complex after internal protease cleavage similar to the
interactions thought to be involved in tetramer formation. Analysis of
hydrodynamic radii of recombinant peptides containing differing numbers
of motifs showed that a single motif had a Stokes radius of 2.35 nm,
while each additional motif added only 0.85 nm to the Stokes radius.
This is the first direct demonstration that spectrin's
flexibility arises from regions between each triple helical motif
rather than from within the segment itself and suggests that current
models of inter-motif connections may need to be revised.
The membrane skeleton of the human erythrocyte consists of a
network of proteins that associates with the inner surface of the cell
membrane and imparts remarkable structural integrity and flexibility to
circulating erythrocytes. Spectrin is the major structural component of
this specialized submembranous protein network. The basic functional
unit of spectrin is a heterodimer formed by side-to-side, antiparallel
association of a 280-kDa subunit with a 246-kDa
subunit.
Spectrin dimers associate head-to-head to form tetramers, the
predominant form of spectrin in the membrane skeleton. These tetramers
cross-link short actin oligomers, an association modulated by band 4.1,
to form a dynamic two-dimensional submembrane latticework. Other
associated proteins include: ankyrin, adducin, calmodulin, tropomyosin,
tropomodulin, and band 4.9 (for reviews, see Bennett and Gilligan
(1993), Delaunay and Dhermy(1993), Luna and Hitt(1992), Winkelmann and
Forget(1993), and Lux and Palek(1995)).
Electron microscopy of
spectrin dimers shows flexible 100-nm long rod-like molecules with
strong lateral association of the subunits near the physical ends of
the rods and weak associations in the central region (Shotton et
al. 1979). In contrast, dimers in situ are only about 30
nm in length (Ursitti et al., 1991). This ability of the
spectrin molecule to shorten and extend as well as its flexibility are
attributed to the series of homologous 106-residue segments or motifs
initially identified by partial peptide sequence (Speicher and
Marchesi, 1984) and confirmed by complete sequencing of cDNAs for the
subunit (Sahr et al., 1990) and
subunit
(Winkelmann et al., 1990). Analysis of spectrin motif
conformational phasing (the boundaries for complete folding units)
using recombinant proteins established the starting point of properly
folded spectrin motifs at approximately positions 26-30 of the
original sequence alignment (Winograd et al., 1991). Yan et al.(1993) recently determined the crystal structure of the
14th segment of Drosophila
-spectrin, which directly
confirmed the phasing predicted from the recombinant peptide phasing
experiments as well as the triple helical conformation of the basic
106-residue spectrin motif.
Previous studies of lateral association
of and
subunits (Morrow et al., 1980; Sears et
al., 1986; Yoshino and Minari, 1991; Speicher et al.,
1992) used mild trypsin digestion to dissect spectrin into a
reproducible pattern of intermediate-sized peptides. The latter study
used a HPLC (
)gel filtration assay to analyze association of
tryptic peptides with complementary spectrin subunits. These analyses
showed that dimer assembly occurs very rapidly (within seconds) and
that dimerization required a specific region at the tail end of the
subunits represented by the tryptic
V and
IV domains
(Speicher et al., 1992). These tryptic domains include most or
all of the repetitive segments
19-21 and
1-4.
Interaction of these regions is apparently the initial step of dimer
assembly, which is followed by subsequent lateral association of
additional
and
motifs. A mutation in
-spectrin in this
region has been identified,
, that affects dimer
assembly (Alloisio et al., 1991; Wilmotte et al.,
1993; Randon et al., 1994). When this mutation is present
along with an elliptocytosis mutation on the same chain, symptoms of
the elliptocytosis mutation are often silent or mitigated since the
mutation will decrease incorporation of the
mutated chain onto the membrane. In contrast, elliptocytosis mutations
on the opposite allele from the
mutation enhance
incorporation of
subunit carrying the elliptocytosis mutation
onto the membrane (Garbarz, 1994; Wilmotte et al., 1993). The
ability of the
mutation to affect dimer assembly
highlights the functional and clinical importance of the dimer
nucleation region.
In the current study, we purified and extensively
characterized a series of nucleation region recombinant peptides
for proper polypeptide chain folding, dimer binding affinity, and
hydrodynamic properties. These analyses show that the minimum
peptide for dimer assembly contained the first two homologous motifs,
but not the actin binding domain, and each additional motif further
increased dimer binding affinity. Analysis of hydrodynamic radii of
these recombinant peptides provided the first direct demonstration that
spectrin's flexibility apparently resides in the connecting
region between triple helical motifs rather than within the segment
itself.
Five -spectrin nucleation site clones
and one
-spectrin nucleation site clone were produced for this
study. The specific oligonucleotide primers used are listed in Table 1. Initially, the start site (phasing) for a clone
encompassing the first four homologous
motifs,
1-4,
used the codon for amino acid residue 301, which corresponds to the
phasing reported by Winograd et al. (1991). Further analysis
of the potential start site of this
1-4 peptide led to the
production of another clone,
1-4
, which
contains eight additional codons at the N terminus of the expressed
peptide. Subsequent truncations of full motifs on the C-terminal and
N-terminal ends of the
1-4
recombinant were
prepared as described in Table 1.
The entire -spectrin
nucleation site, encompassing repetitive motifs
18-21, was
designed essentially as described above with the exception that the
nucleotide sequence contained a BamHI restriction enzyme site.
Therefore, the oligonucleotides used for polymerase chain reaction were
designed to contain a BglII site both at the 5` and 3` ends.
This restriction enzyme creates the same overhang as BamHI
thus allowing cloning into the BamHI site of pGEX-2T. A BglII site near the 3` end of the region to be amplified was
apparently not changed by altering the spectrin sequence in the 3`
primer. This resulted in utilization of the stop codon of the pGEX-2T
vector. A single motif
recombinant,
1 (residues
50-158), was prepared as described previously (Kotula et
al., 1993).
All expression plasmids were transformed into the
DH5 strain of Escherichia coli. Each construct was
completely sequenced to verify the integrity of the recombinant
vectors. The
- and
-spectrin cDNA clones were kindly provided
by Dr. Bernard Forget (Yale University, New Haven, CT).
Figure 1:
Recombinant spectrin peptides. A, diagrammatic correlation of recombinant nucleation site
peptides with the 106-residue repetitive spectrin motif. The most
commonly used alignment of homologous spectrin sequences (Speicher and
Marchesi, 1984), as indicated by the numbers at the top of the
figure, is correlated with the three helices designated
``A,'' ``B,'' and
``C'' that form the triple helical conformational
unit defined by crystallography (Yan et al., 1993). Spaces
between the three helices represent turn regions. The recombinant
peptides are illustrated by horizontal lines labeled with the
repetitive motif number in the right margin (see panel C for
location of these motifs within the spectrin dimer). Non-spectrin amino
acids introduced at the ends of recombinant peptides by construction of
expression vectors are indicated using single letter code. The phasing
of all recombinant peptides is as defined by the crystallographic model
and prior analysis of recombinant peptides (Winograd et al.,
1991) except for the ``+'' series of peptides.
This series of peptides begins 8 residues earlier than the normal
conformational motif. The C-terminal end of the
1
(*),
1-2
(
), and
1-3
(
) peptides are marked as
indicated. B, SDS gels of
-monomers and recombinant
peptides after cleavage from the GST fusion moiety and repurification.
The samples are: lanes 1 and 2 (4 µg/lane),
-spectrin monomer and
18-21, respectively, on a 7%
Laemmli gel; lanes 3-7 (2 µg/lane),
1-4
,
1-4,
1-3
,
2-4, and
1-2
, respectively, on a 10% Laemmli gel;
and lane 8 (2 µg/lane),
1
on a
5-15% linear gradient Tricine gel. C, arrangement of the
structural motifs in an anti-parallel spectrin dimer. The
subunit
is comprised of an actin binding domain (ABD), 17 homologous
motifs (numbered rectangles), and a small non-homologous
phosphorylated C-terminal domain (solid squiggle). The
subunit is comprised of an N-terminal partial and 20 full homologous
motifs (motifs 1-9 and 11-21), an SH-3 type motif (motif
10), and a non-homologous C-terminal region consisting primarily of two
EF-hand type motifs (diamonds).
In addition to
the recombinant constructs shown in Table 1and Fig. 1A, the 1
peptide was
isolated as a proteolytic by-product of the
1-2
construct. During purification of the
1-2
peptide, a highly specific cleavage product was observed due to
cleavage at residue 425 (see Fig. 1A) as defined using
MALDI mass spectrometry which yielded an experimental mass after
removing the GST moiety by thrombin cleavage of 15,688 Da (data not
shown). This proteolytically produced peptide contained the entire
1
motif with only a few additional residues on
the C-terminal end (see Fig. 1A), was native as shown
by circular dichroism and gel filtration, and was easily separated from
the parent
1-2
peptide by gel filtration.
All recombinant peptides were expressed in bacterial cells as fusion
proteins with glutathione S-transferase (GST), which was
cleaved and separated from the recombinant proteins prior to use for
functional studies. The purity of all peptides was >95% (Fig. 1B). The N termini of the cleaved peptides were
confirmed by N-terminal sequence analysis and masses were confirmed by
MALDI mass spectrometry (data not shown). The 1-4 construct
was difficult to cleave from the GST moiety and a substantial amount of
a secondary cleavage product was produced (lane 4,Fig. 1B). N-terminal sequence analysis of this
smaller peptide showed that it resulted from a secondary cleavage after
Arg
, which is located in the turn between helices B and C
of motif
1. The full-length
1-4 peptide and its
cleavage product could not be separated by either ion exchange or gel
filtration chromatography due to their similar size, charge, and other
physical properties.
Figure 2:
Correlation of the elongated spectrin and
-actinin nucleation site motifs with the more common 106-residue
motifs and crystallographic structure. A, the phasing and
conformation of a 106-residue motif determined by crystallography (see Fig. 1A) as illustrated at the top of the panel
(helices A, B, and C) was used to align and compare spectrin nucleation
site motifs (
18-21 and
1-4) and the spectrin-like
motifs of
-actinin that are responsible for
-actinin dimer
formation (A1-4). Insertions required to fit these longer
sequences into the more common 106-residue motif are shown as underlined sequences above the main sequence and the insertion
site is indicated with an arrowhead. The positions of
insertions were determined using the program ALIGN. Residues that are
identical in at least two of the three aligned sequences are shaded. Prolines are bold and underlined. Numbers on the right are the number of identities
between the indicated pairs of motifs. B, relationship of the
predicted nucleation site phasing with cleavage sites of spectrin and
-actinin produced by limited proteolysis. Mild cleavage of
-actinin with chymotrypsin produces a 55-kDa fragment (C55K) as initially shown by Imamura et al.(1988) and
confirmed by us. One of the first cleavages of
-spectrin with
trypsin removes the ABD domain by cleavage at residue 292 and the
earliest observed fragments that contain the
nucleation site are
T74K and T46K. Alignment of the N-terminal sequences of these peptides
are shown and the arrow indicates the predicted nucleation
site phasing shown in panel A and used to construct the
series of recombinant peptides (see
text).
Based on the
alignment in Fig. 2, it was hypothesized that the first motif
should have an 8-residue insertion in helix A relative to the more
typical 106-residue motif, since the three C-terminal -actinin
motifs (A2, A3, and A4) and their most homologous spectrin counterparts
(
2,
20, and
21, respectively) all contained an extra 8
residues in this region. This prediction of a start site for the first
motif that is 8 residues earlier than the initial
1-4
recombinant compares favorably with the observed mild protease cleavage
sites for both
-actinin and
-spectrin as shown in Fig. 2B. In addition, although various start sites have
been reported for the
-actinin motif based on alignments of
sequences, the revised start site presented here for the first motif of
spectrin and
-actinin agrees with the recent phasing analysis of
-actinin using recombinant peptides reported by Gilmore et
al.(1994).
To further evaluate the phasing of the first
motif, a
1-4
recombinant peptide, which
started at residue 293 compared with residue 301 for the
1-4
peptide, was produced and analyzed in parallel with the
1-4
peptide. Thrombin cleavage of the
1-4
and
1-4 fusion proteins differed markedly at physiological ionic
strength. The
1-4
fusion protein was
efficiently cleaved without production of secondary cleavage products.
In contrast, under the same conditions, <50% of the
1-4
protein was cleaved and a prominent secondary cleavage product was
formed (data not shown).
Figure 3:
CD
spectra of -monomer and representative recombinant peptides.
-
-,
-monomer (0.47 mg/ml); -,
1-4
(0.33 mg/ml); - - - -,
1-3
(0.47 mg/ml); and - -,
1-2
(0.38 mg/ml). All proteins were
dialyzed into isotonic buffer and protein concentrations were
determined by quantitative amino acid analysis prior to CD
measurements. Mean residue ellipticity [
]
is expressed in degree
cm
/dmol.
The
Stokes' radii of the -spectrin nucleation site recombinant
peptides and a single motif
subunit peptide,
1 (residues
50-158), were determined by HPLC gel filtration using standard
proteins with known Stokes' radii to calibrate the column. The
observed hydrodynamic radii were linearly related to the number of
repetitive motifs as shown in Fig. 4. A single repetitive
segment, either
1
or
1, has a Stokes'
radius of 2.35 nm and each additional motif increases the molecular
size by only 0.85 nm. The Stokes' radius of the
1-4
peptide falls below the line created by the other nucleation site
peptides and was not included in the linear regression calculation.
This smaller Stokes' radius is indicative of a more compact
molecular shape for this improperly phased
1-4 peptide,
however, it did have normal helicity as determined by circular
dichroism.
Figure 4:
Hydrodynamic properties of recombinant
-spectrin nucleation site peptides. The Stokes' radii of
-spectrin recombinant peptides were determined by HPLC gel
filtration. Peptides include
1-4 (
),
1-4
(+),
1-3
(+),
2-4 (
),
1-2
(+),
1
(+), and
1 (
)
with duplicate determinations shown for
1-4
. The
1 motif peptide (residues
50-158) is described in Kotula et al.(1993). The more
compact
1-4 peptide was not included in the linear
regression plot. Slope = 0.85, y intercept =
1.5, R
= 0.9994.
Figure 5:
Binding of -spectrin recombinant
peptides to
-monomers. Purified recombinant peptides were mixed
with
-spectrin monomer and incubated at 0 °C for 25 min before
separation by HPLC gel filtration. In control experiments, an equal
volume of buffer was substituted for
-monomer. A, solid lines are chromatograms of association experiments where
500 pmol of the recombinant
peptide indicated in the panel was
mixed with 500 pmol of
-monomer. Dashed lines are
chromatograms of the recombinant peptide alone (offset by +10 mA
units for clarity). B, SDS gel of the bound (B) and
unbound (U) fractions from the binding experiments shown in panel A (
-monomer portion of gel not
shown).
Binding affinities of the recombinant peptides
with
-monomers are summarized in Table 2. The importance of
N-terminal phasing for the first
motif is illustrated by the
observation that the
1-4 peptide has nearly a 10-fold lower
affinity for
-monomers compared with the 8-residue longer
1-4
peptide. The minimum dimerization site
contains the first two motifs, which has a K
of
about 230 nM. Each additional motif contributes to the
affinity of the complex apparently through low affinity lateral pairing
of additional motifs and a 4-motif nucleation site peptide has a K
of about 10 nM.
The lateral
association of recombinant peptides with
-monomers is
readily reversible. As shown in Fig. 6, both the high affinity
1-4
peptide and the lower affinity
1-2
peptides can compete with each other
for binding to
-spectrin monomers.
1-2
was also able to compete with
1-3
for
binding to
-spectrin (data not shown).
Figure 6:
Competition of -spectrin nucleation
site peptides for association with
-monomers. Dashed lines are control experiments where
-monomers and a
nucleation site peptide (200 pmol each) were incubated 25 min at 0
°C and separated by HPLC gel filtration. Solid lines are
competition experiments where duplicates of the above controls received
a 5-fold molar excess of a different
nucleation site peptide and
were incubated for an additional 25 min prior to HPLC separation. See Fig. 5A for additional details. Upper panel: -
- - -,
-monomer +
1-4
; -,
-monomer +
1-4
+ 5
1-2
. Lower panel, - - - -,
-spectrin +
1-2
, -,
-spectrin +
1-2
+ 5
1-4
.
Further analysis of these samples by Tricine gel electrophoresis detected a band at about 8 kDa. Similarly, samples that were initially separated by HPLC gel filtration still contained the 8-kDa peptide. MALDI mass spectrometry of the peptide mixture confirmed that the 8-kDa (observed mass = 8,733.8 Da versus expected mass for GS + 301-374 = 8,725 Da) and 43-kDa fragments (observed mass = 43,398.3 Da versus expected mass for 375-743 = 43,379 Da) were produced by a single protease cleavage at residue 374 and that the 43-kDa fragment had an intact C-terminal. Since the expected 0.1% error of this technique is less than a single amino acid residue mass, this method reliably defines the C-terminal boundary of proteins with known sequences when the N-terminal has been determined by sequence analysis.
A previous report from our laboratory using peptides produced
by mild proteolysis showed that spectrin dimers assembled like a zipper
with initiation of the process occurring near the tail end (actin
binding end) of the molecule (Speicher et al., 1992). In the
present study, we further characterized dimer nucleation using
recombinant peptides. The primary structure and conformational
integrity of these recombinant peptides were confirmed by full-length
DNA sequencing, N-terminal sequencing of the cleaved peptide, mass
spectrometry, and circular dichroism measurements to ensure that the
peptides were free of polymerase chain reaction-based mutations and
properly folded. Therefore, the observed differences in dimer assembly
properties of the N-terminal and C-terminal truncations of the
-spectrin nucleation site represent functionally important
findings.
The minimum peptide capable of dimerizing to the
subunit contains the first two homologous motifs (
1 and
2), and each additional motif (
3 and
4) increases the
binding affinity approximately 5-fold, apparently by forming additional
lower affinity lateral associations with a complementary motif in the
-monomer (Table 2). Although direct binding affinity
measurements have only been made here on recombinant peptides with
lengths up to 4 motifs, dimer affinity continues to increase with each
additional motif as it laterally pairs with its complementary partner
throughout the length of the two subunits (Speicher et al.,
1992). This size dependent increase in dimerization affinity is
apparently due to formation of additional lateral associations outside
the first 4 motifs since preferential dimerization of larger peptides
is not observed when either nucleation site 4-motif recombinant fusion
protein (GST-
1-4
or GST-
18-21)
is used instead of the complementary monomer (Speicher et
al.(1992) and data not shown).
The 1 motif is required for
dimerization, but is insufficient for high affinity association as
shown by the loss of dimer formation capacity of the native
2-4 and the
1
recombinant peptides (Fig. 5). In addition, these experiments showed that the precise
phasing (starting point) of the
1 motif had a critical effect on
both binding affinity ( Fig. 5and Table 2) and molecular
shape (Fig. 4). The appropriate N-terminal boundary of this
motif is different from the phasing that applies to the more common
106-residue spectrin-type motif.
This altered start site for the
1 motif and its structural and functional importance had not been
previously identified. The presence of an additional 8 amino acids
relative to the 106-residue motifs had been proposed near the beginning
of erythrocyte spectrin motifs
20,
21, and
2 (Sahr et al., 1990; Winkelmann et al., 1990) and near the
beginning of Drosophila spectrin motifs
20,
21, and
2 (Viel and Branton, 1994). The corresponding start site for the
closely related first
-actinin motif had not been clearly defined,
with reported possibilities ranging from
-actinin residue 245 to
266 (Baron et al., 1987; Imamura et al., 1988;
Blanchard et al., 1989). The difficulty encountered in
cleaving a
1-4 fusion protein using the phasing defined for
106-residue spectrin-type motifs and further consideration of the
locations for mild protease cleavage sites for
-spectrin and
-actinin (Fig. 2B) suggested that both the first
motif and the first
-actinin motif may have an extra 8
residues in the first helix as suggested by the alignment in Fig. 2A. The resulting improved thrombin cleavage of
the fusion protein, loss of secondary cleavage site, larger solution
molecular shape, and nearly 10-fold higher binding affinity of a
recombinant protein with an additional 8 residues on the N-terminal
supported this hypothesis. It is particularly striking that the
dimerization affinity of the 8-residue shorter
1-4 peptide
has an order of magnitude lower binding affinity for
-monomers
compared with the
1-4
peptide and its
affinity is even lower than the
1-3
peptide. The motif phasing determined experimentally for the
1 motif and inferred for the first
-actinin motif in this
study is consistent with the
-actinin motif start site recently
determined by Gilmore et al. (1994) using recombinant
peptides.
The nomenclature for spectrin motifs used in this study is
as described by Winkelmann et al.(1990) where only homologous
motifs are numbered from 1 to 17 (see Fig. 1C). A
recent publication describing dimer assembly of Drosophila spectrin (Viel and Branton, 1994) uses an alternate nomenclature
where the actin binding domain is designated as the first segment of
-spectrin. Although each nomenclature has its merits, the
nomenclature used by Winkelmann et al.(1990) for human
erythroid spectrin has been more frequently cited and is used here.
The largest discrepancy between the present study and the
qualitative evaluation of Drosophila spectrin dimer assembly
(Viel and Branton, 1994) is that the non-homologous regions after the
21 motif (EF-hand motifs) and before the
1 motif (actin
binding motif) of Drosophila spectrin were found to be
required for high affinity dimer assembly. The corresponding regions of
human erythroid spectrin are clearly not required for dimer assembly
since the
1-4
recombinant could associate
with either
-spectrin monomers or an
18-21 recombinant
protein with a K
of approximately 10 nM and intact
-monomers bind to the
18-21 recombinant
with a similar affinity. Although these experiments do not rule out a
direct interaction of the
EF-hand motifs with the
-spectrin
actin binding domain, this potential interaction is clearly not
required for initiation of dimer assembly as demonstrated in this
study. In addition, the possible interaction between the EF-hand motifs
and the actin binding domain would be expected to be a low affinity
interaction since the actin binding domain is quickly cleaved from
dimers with trypsin and does not remain covalently bound to the intact
subunit (Speicher et al.(1992) and data not shown). It
is particularly surprising that the Drosophila
1-4
288 peptide is inactive since it corresponds
closely to our
1-3
recombinant, which has a K
of about 38 nM. These differences could
reflect a species and/or tissue-specific isoform difference since Drosophila spectrin is more closely related to the human brain
spectrin (fodrin) isoform than to erythroid spectrin. Also, the Drosophila recombinant peptides, which were produced in a
reticulocyte lysate system and not purified to homogeneity, had unknown
conformational integrity. Some nonfunctional Drosophila peptides may not have folded properly or were not stable enough to
retain binding activity in the presence of SDS used in the
immunoprecipitation buffer. It should be noted that Lombardo et
al.(1994) cited substantial difficulties in preparing stable,
native peptides of brain
-fodrin that contain only a portion of
the actin binding domain. Since the present study shows that the
phasing of the
1 motif has a dramatic effect on dimer binding
affinity, it is most likely that some Drosophila
peptides were too short and other peptides may not have correctly
folded as suggested by the observations of Lombardo et
al.(1994).
Thrombin cleavage of the improperly phased
1-4 fusion protein led to the interesting observation that
interactions between helices within a single conformational motif are
strong enough to retain noncovalent complexes during extensive dialysis
or gel filtration chromatography (``Results''). Similar
noncovalent associations within a triple helical motif were observed
between adjacent peptides produced by mild trypsin treatment from both
the
(DiPaolo et al., 1993) and
subunits (Speicher et al., 1992) suggesting that, as a general rule, inter-helix
interactions within triple helical spectrin-type motifs are very high
affinity interactions. These helix A-B
C interactions may be
similar to the interaction between incomplete motifs of the
and
subunits that form the tetramer binding site (Tse et
al., 1990; Speicher et al., 1993; Kotula et al.,
1993; Parquet et al., 1994; Kennedy et al., 1994).
Comparison of Stokes radii of recombinant proteins used in this
study provided a unique opportunity to evaluate the relative
contributions of individual motifs and the connecting regions between
motifs to the molecular shape in solution. This comparison is of
particular interest since the recent crystallographic model of a single
spectrin motif predicts that adjacent motifs are linked by a single
long helix formed by continuing the C helix of the first motif into the
A helix of the next motif (Yan et al., 1993). In this model,
the substantial molecular flexibility associated with spectrin might
arise from dynamic rearrangements within the triple helical bundle
and/or could involve a non-apparent disruption of the helix between
motifs. Alternatively, adjacent motifs might be connected by a
flexible, non-helical linking region as suggested by some earlier
models. Measurement of the Stoke's radii of two single motif
peptides, the non-nucleation site 1 peptide and the nucleation
site
1 peptide, showed a disproportionately high contribution of
the first motif to the molecular shape (2.35 nm), while each additional
motif contributes only about 0.85 nm to the molecular size. These
results strongly suggest that individual motifs are relatively rigid in
solution as might be expected for triple helical bundles with strong
inter-helix interactions (see above). The substantially smaller and
uniform incremental increase with each additional motif suggests that
there is substantial flexibility in the connection or interface between
motifs that allow the motifs to pleat and fold as suggested by models
(Bloch and Pumplin, 1992) derived from electron microscopic evidence
(Shotton et al., 1979; Byers and Branton, 1985; Shen et
al., 1986; Liu et al., 1987; Ursitti et al.,
1991). These data suggest that the current model of spectrin structure
where adjacent segments are connected by one long helix should be
re-evaluated.