Interactions of the alpha -Spectrin N-terminal Region with beta -Spectrin
IMPLICATIONS FOR THE SPECTRIN TETRAMERIZATION REACTION*

Lisa Cherry, Nick Menhart, and Leslie Wo-Mei FungDagger

From the Department of Chemistry, Loyola University of Chicago, Chicago, Illinois 60626

    ABSTRACT
Top
Abstract
Introduction
References

Spectrin of the erythrocyte membrane skeleton is composed of alpha - and beta -spectrin, which associate to form heterodimers and tetramers. It has been suggested that a fractional domain (helix C) in the amino-terminal region of alpha -spectrin (Nalpha region) bundles with another fractional domain in the carboxyl-terminal region of beta -spectrin (Cbeta region) to yield a triple alpha -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 alpha -spectrin fragments spanning only the Nalpha region (lacking the dimer nucleation site) and quantitatively studied their interaction with beta -spectrin. We found that a majority of the interactions were localized, as expected, in the Nalpha -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 alpha - and beta -spectrin undoubtedly play a significant role in tetramerization, regions distal to the Nalpha -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 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, alpha -spectrin (280 kDa) and beta -spectrin (246 kDa), which associate to form alpha beta heterodimers (2, 3), which then associate to form the biologically relevant (alpha beta )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).

Amino acid (12) and cDNA (13) sequence analyses show that both alpha - and beta -spectrin are largely composed of multiple homologous motifs of about 106 amino acid residues. These sequence motifs are suggested to fold into triple alpha -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-alpha -helical bundle structure is thought to be stabilized by the sequestration of the hydrophobic faces of each helix to the interior of the bundle.

The initial, antiparallel, side-by-side association of alpha - and beta -spectrin to form alpha beta dimers occurs at the amino terminus of beta -spectrin and the carboxyl terminus of alpha -spectrin, with high affinity (Kd ~ 10 nM) (20). This site is referred to as the dimer nucleation site. The rest of the alpha beta 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 alpha -spectrin (Nalpha region)1 and the carboxyl-terminal region of beta -spectrin (Cbeta region) have been shown to encompass fractional 106-amino acid sequence motifs (23, 24). The Nalpha region contains a nonhomologous sequence, followed by a sequence homologous to that of the third alpha -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 alpha -spectrin sequence. We will refer to this fractional motif region as Nalpha -helix C. It should be noted that there is no direct experimental evidence that this fractional domain is or is not entirely alpha -helical. This designation is made solely on the basis of primary sequence homology (12). Similarly, the Cbeta region contains a nonhomologous segment and a longer fractional motif homologous to the sequence of the first two alpha -helices in a complete motif.

During the association of two dimers to form tetramers, it has been suggested that the major interaction involves the bundling of the Nalpha -helix C with the two helices of the Cbeta region to yield a triple alpha -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 Nalpha -helix C region or the Cbeta region with the two helices (10, 11, 26-29). In most cases, it has been found that these spectrin mutants render unstable tetramers.

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 alpha beta dimer, the longer alpha -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 Nalpha /Cbeta 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 Nalpha -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, alpha -spectrin is shown shaded, while beta -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 alpha beta dimers form hairpin loops (A), which are stabilized by an alpha beta complex formation involving helical bundling of the Nalpha -helix C and the helices in the Cbeta region. These helical bundles need to be broken to form transient intermediates with "sticky ends" (B) before they can reassociate to form the (alpha beta )2 tetramer (C). Contributions to the energetics of the process include not only the alpha  and beta  association (helical bundling) energy, Delta Gbundle, but also possibly the energy needed to bend the alpha -spectrin extension back on itself, Delta Ghairpin, as well as lateral interactions between the adjacent alpha -spectrin regions of the tetramer, Delta Galpha -lateral. The overall energetics (Delta Gtetramer:dimer) for this model is thus independent of Delta Gbundle, with Delta Gtetramer:dimer = 2Delta Ghairpin - 2Delta Gbundle + 2Delta Gbundle + Delta Galpha -lateral = 2Delta Ghairpin + Delta Galpha -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 alpha beta 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 alpha -spectrin peptides spanning only the Nalpha region (lacking the dimer nucleation site) and studied their interaction with beta -spectrin quantitatively. These systems eliminated the nucleation interaction and the hairpin structure in alpha -spectrin to allow us to focus on only the helical bundling interaction. We found that a majority of the interactions in our alpha -spectrin peptides with beta -spectrin were attributed to helical bundling in the Nalpha -helix C region. More importantly, the temperature and ionic strength dependence of our alpha -peptides binding to beta -spectrin was different from that in intact spectrin. We suggest that, although the regions involving the putative helical bundling in alpha - and beta -spectrin undoubtedly play a significant role in tetramerization, regions distal to the Nalpha -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.

    EXPERIMENTAL PROCEDURES

Preparation and Analysis of beta -Spectrin and of alpha -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). beta -spectrin was obtained from spectrin dimer as before (21), except that beta -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 alpha -peptides, consisting of the first three complete triple alpha -helical structural domains (residues 52-368), as well as (a) the entire leading nonhomologous region (residues 1-20) and the Nalpha -helix C region (residues 21-51) to give peptide Spalpha -(1-368), (b) half of the nonhomologous region and the entire Nalpha -helix C region (Spalpha -(12-368)), (c) only the Nalpha -helix C region (Spalpha -(22-368)), (d) half of the Nalpha -helix C region (Spalpha -(37-368)), and (e) no Nalpha -helix C region (Spalpha -(52-368)). In addition, we also prepared similar peptides with only one (Spalpha -(1-156)) or two (Spalpha -(1-262)) complete structural domains.

The cDNA encoding the amino terminus of alpha -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 Spalpha -(1-368), Spalpha -(1-262), Spalpha -(1-156), and Spalpha -(52-368) were prepared as described previously (25, 32). The genes encoding for the peptides Spalpha -(12-368), Spalpha -(22-368), and Spalpha -(37-368) were derived from the gene for Spalpha -(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-alpha -Spectrin Peptides-- Interaction of alpha -peptides with the beta -spectrin was examined by competition experiments using unlabeled peptide to displace 125I-labeled peptide bound to beta -spectrin. Iodination of alpha -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 alpha -peptides with beta -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, Spalpha -(1-446) (25), and examined it for binding to spectrin dimer and beta -spectrin. Wells of a microtiter plate (Immulon-4 from Dynatech, Chantilly, VA) were coated with spectrin dimer or beta -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-Spalpha -(1-446) (~0.03 µM; ~50,000 cpm) was then added to these wells. Unlabeled Spalpha -(1-446) (10 µM) was added to some wells to compete with the 125I-Spalpha -(1-446), followed by washing with the isotonic buffer containing 6% milk. The amount of 125I-Spalpha -(1-446) remaining in each well was measured with a gamma -counter (ICN Micromedic Systems).

To ensure sample equilibrium and to optimize the 125I signal, the on and off time constants for this assay (tau on and tau off) of an alpha -peptide (Spalpha -(1-368)) were also checked. A constant amount of 125I-Spalpha -(1-368) was added to all wells, and the incubation period was varied from 0.5 to 50 h. The tau 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-alpha -peptide and beta -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 tau off ~10 min. The remaining alpha -peptide (~80%) came off much more slowly with a tau off > 6 h. Therefore, there was not a significant disturbance of equilibrium during washing, which was accomplished in <1 min.

Binding Affinity Assay-- beta -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-alpha -peptide (typically about 100,000 cpm, corresponding to about 0.05 µM), was added to the wells for interactions with the immobilized beta -spectrin. Concurrently, unlabeled alpha -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 beta -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 ([alpha ]). 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 beta -spectrin) were determined according to the equation (35, 36),
<UP>cpm<SUB>bound</SUB></UP>=A<SUB>0</SUB>+<UP>cpm<SUB>max</SUB></UP>/(1+[&agr;]/<UP>IC</UP><SUB>50</SUB>) (Eq. 1)
where cpmmax was the amount of labeled peptide bound in the absence of unlabeled alpha -peptide, and A0 was the nonspecific binding background signal (typically <10% of cpmmax). It was assumed that the beta -spectrin concentration in this assay was much less than [alpha ] and IC50. Generally, 3-5 runs were done on at least two separate preparations of beta -spectrin and alpha -peptides, and IC50 values were averaged.

For homologous competition experiments, the 125I-labeled and unlabeled peptides were of the same type (e.g. 125I-Spalpha -(1-368) and unlabeled Spalpha -(1-368)). For heterologous competition experiments, different unlabeled peptides were used to compete with the binding of 125I-Spalpha -(1-368) to beta -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 alpha -peptides with different binding affinities toward the beta -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 Spalpha -(1-368) toward beta -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.

    RESULTS

Protein Analysis-- The identity of beta -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 alpha -peptides were ~18 kDa for Spalpha -(1-156), 31 kDa for Spalpha -(1-262), 43 kDa for Spalpha -(1-368), 41 kDa for Spalpha -(12-368), 40 kDa for Spalpha -(22-368), 39 kDa for Spalpha -(37-368), and 37 kDa for Spalpha -(52-368). All seven peptides were also analyzed by electrospray mass spectrometry and Edman degradation of the amino terminus. Electrospray ionization mass spectrometry of Spalpha -(1-156), Spalpha -(1-262), Spalpha -(1-368), Spalpha -(12-368), Spalpha -(22-368), Spalpha -(37-368), and Spalpha -(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 Spalpha -(1-156), Spalpha -(1-262), Spalpha -(1-368), Spalpha -(12-368), Spalpha -(22-368), Spalpha -(37-368), and Spalpha -(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 Spalpha -(37-368) peptide appeared to exhibit slight aggregation, since the ratio was about 1.2. The alpha -peptides were found by SDS-polyacrylamide gel electrophoresis to be at least 90% pure.

The alpha -helical contents from CD analysis of Spalpha -(1-156), Spalpha -(1-262), Spalpha -(1-368), Spalpha -(12-368), Spalpha -(22-368), Spalpha -(37-368), and Spalpha -(52-368) were 57, 73, 76, 74, 75, 69, and 76%, respectively, based on a value of 36,000 for 100% alpha -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 Spalpha -(1-446), the amount of 125I-Spalpha -(1-446) bound to spectrin dimer wells (457 ± 148 cpm for wells with Spalpha -(1-446) and 502 ± 117 cpm for wells without, n = 3) did not vary, whereas the amount of 125I-Spalpha -(1-446) bound to beta -spectrin wells was significantly reduced (1246 ± 373 cpm for wells without and 104 ± 25 cpm for wells with). These results showed that Spalpha -(1-446) bound specifically to beta -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 alpha - and beta -spectrin (21). Thus, the two helices in beta -spectrin in immobilized dimers were not available for bundling with the helix in Spalpha -(1-446). The 125I activities found in the spectrin dimer wells, with or without unlabeled Spalpha -(1-446), probably reflected the nonspecific binding of 125I-Spalpha -(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 beta -spectrin coated the wells of the titer plate differently, and thus different amounts of residual nonspecific binding of 125I-Spalpha -(1-446) to wells (502 ± 117 cpm for spectrin dimer and 104 ± 25 cpm for beta -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) Spalpha -(1-446), further demonstrating binding specificity toward beta -spectrin and not other proteins.


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Fig. 2.   Binding specificity. Microtiter plate wells were coated with either spectrin dimer or beta -spectrin. 125I-Spalpha -(1-446) was added in either the absence (hatched bars) or presence of 10 µM unlabeled Spalpha -(1-446) (solid black bars), and the amount bound in each well in isotonic buffer at 4 °C was measured in quadruplicate with the S.E. indicated by error bars. No specific (competed by unlabeled Spalpha -(1-446)) binding was observed for spectrin dimer, but specific binding was observed for beta -spectrin (12-fold more binding in the absence of unlabeled peptide). The lack of binding to the spectrin dimer may be attributed to the fact that titer plate-bound spectrin dimer was "locked" into the "hairpin" type conformation, as shown in Fig. 1, and no binding was expected. The 125I activities found in the spectrin dimer wells, with or without unlabeled Spalpha -(1-446), probably reflected nonspecific binding of 125I-Spalpha -(1-446) to the 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 beta -spectrin coated the wells of the titer plate differently, and thus different residual nonspecific binding of 125I-Spalpha -(1-446) to wells was observed. The amount of 125I-Spalpha -(1-446) bound to beta -spectrin was lower than typical values obtained in these assays, probably due to inhomogeneity of microtiter plates.

Binding Affinity-- For homologous displacement assays, Fig. 3A shows that all peptides except Spalpha -(37-368) and Spalpha -(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 alpha -spectrin sequence, i.e. Spalpha -(1-156), Spalpha -(1-262), and Spalpha -(1-368), displayed IC50 values of about 0.3 µM, with values for Spalpha -(1-368) being the lowest (0.24 ± 0.05 µM) and Spalpha -(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 Spalpha -(1-368) toward beta -spectrin was larger than that of Spalpha -(1-156).


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Fig. 3.   Solid phase binding assays. A, homologous peptide displacement assays were conducted by 125I labeling each peptide (black-square, Spalpha -(1-156); bullet , Spalpha -(1-262); black-triangle, Spalpha -(1-368); black-down-triangle , Spalpha -(12-368); black-diamond , Spalpha -(22-368)) and competing for its binding to microtiter plate-bound beta -spectrin by unlabeled peptide. Lines represent the fit of the experimental points to Equation 1. Data shown were normalized to the value bound in the absence of unlabeled peptide, typically ~10,000 cpm. Spalpha -(1-156), Spalpha -(1-262), and Spalpha -(1-368) displayed similar IC50 values of 0.2-0.3 µM. The peptides beginning at either residue 12 or 22 (Spalpha -(12-368) and Spalpha -(22-368)) showed reduced IC50 values. No data on peptides starting at residue 37 or 52 are shown, since they did not bind specifically to beta -spectrin. B, heterologous binding assays were conducted by using each unlabeled peptide (see A for symbol notations, as well as Spalpha -(37-368) (+) and Spalpha -(52-368) (×) to compete with the binding of 125I-labeled Spalpha -(1-368). IC50 values similar to those in A were obtained. Peptides Spalpha -(37-368) and Spalpha -(52-368) could not compete with Spalpha -(1-368) and therefore did not reduce the amount of bound 125I-Spalpha -(1-368).

                              
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Table I
Binding of various alpha -peptides to beta -spectrin at 4 °C in 10 mM Tris buffer at pH 7.4 with 20 mM KCl and 130 mM NaCl

The peptides with the first 11 or 21 residues removed, Spalpha -(12-368) and Spalpha -(22-368), exhibited a slightly lower affinity toward beta -spectrin, with IC50 values of 0.62 and 0.59 µM, respectively. For peptides with the first 36 or 51 residues removed, Spalpha -(37-368) and Spalpha -(52-368), no binding of 125I-peptides to beta -spectrin was observed.

Heterologous displacement assays were also performed to ensure that the different unlabeled alpha -peptides displaced the 125I-Spalpha -(1-368) peptide. Unlabeled Spalpha -(37-368) and Spalpha -(52-368) did not displace 125I-Spalpha -(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 Spalpha -(1-368) to beta -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 alpha -peptide to beta -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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Fig. 4.   Ionic strength dependence on binding of Spalpha -(1-368) with beta -spectrin at 4 °C. Data are represented as mean values with error bars of one S.D. No marked ionic strength effect was seen over the range 7 mM to 1 M, with the IC50 remaining between 0.17 and 0.28 µM.


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Fig. 5.   Temperature dependence on binding of Spalpha -(1-368) with beta -spectrin in isotonic buffer. A strong temperature dependence was noted, with the binding affinity dropping off rapidly above ambient temperature. Error bars of one S.D. are shown. Over the range 4-30 °C, the data appear to fit an empirical quadratic dependence (solid line) of pIC50 on temperature, (pIC50 = 6.490 + 0.019T - 0.002T2, with T in °C). This may be extrapolated to imply a value of approximately 60 µM at 37 °C.


    DISCUSSION

The association of alpha - and beta -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, alpha I (21), and alpha -recombinant peptides consisting of residues 1-639 (recombinant alpha I) or 1-158 bind beta -spectrin, whereas peptides consisting of residues 31-639 or 28-158 do not bind beta -spectrin (43). These results suggest that some or all of the first 30 amino acid residues in alpha -spectrin are directly involved in the tetramerization site interaction (43), and support the hypothesis that the association involves helical bundling of fractional domains in alpha - and beta -spectrin (23, 24). However, alpha -peptides containing just the fractional domain (residues 7-53) do not bind beta -spectrin and do not appear to have appreciable native structure (43).

We have recently demonstrated cooperative interactions between adjacent structural domains in alpha -spectrin peptides (32). This finding raises the possibility that domains distal to the fractional domain (Nalpha -helix C) may influence the conformation of the Nalpha -helix C region and thus indirectly affect helical bundling and also tetramerization. Quantitative binding parameters, such as Ka, Kd, or IC50 values, of alpha -peptides with different sequences and/or conformations interacting with beta -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 alpha -peptides. These values were in the micromolar range and agreed well with Kd values (at 30 °C) of alpha -peptide consisting of residues 1-158 (43) and of proteolytically digested alpha I fragment (21).

Our assay results at 4 °C in isotonic buffer showed that the binding of Spalpha -(1-156) (peptide with one full domain) or of Spalpha -(1-262) (peptide with two full domains), to beta -spectrin was very similar to that of Spalpha -(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 alpha -spectrin did not appear to affect the helical bundling at the Nalpha /Cbeta region. The removal of either half (Spalpha -(12-368)) or the entire (Spalpha -(22-368)) nonhomologous leader region reduced the affinity by about 2.5 times when compared with Spalpha -(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 beta -spectrin. The majority of the interaction, however, appeared to be mediated by the residues in the Nalpha -helix C region (residues 21-51). Elimination of half of this region (Spalpha -(37-368)) substantially reduced the binding affinity, with IC50 >20 µM. Thus, the dominant binding region in alpha -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 beta -spectrin (43).

Speicher et al. (23) have proposed a "closed" form for the dimer, with a hairpin bend in the alpha -spectrin, the longer piece of the two (alpha  and beta ) subunits, when alpha - and beta -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 alpha -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 Nalpha and Cbeta 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 alpha - and beta -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 Nalpha /Cbeta interactions (Delta Gbundle) but also on other interactions between the two alpha -chains in the region distal to the immediate Nalpha and Cbeta regions (Delta Galpha -lateral) as well as a term related to the flexibility of the alpha -chain (Delta Ghairpin). This scheme predicts that the overall reaction is expected to be energetically neutral with respect to the Delta Gbundle term, since two such Nalpha /Cbeta interactions (helical bundling) are formed in the tetramer, but two are also broken in each of the normally closed (hairpin) dimers. Delta Galpha -lateral contributions have been hinted at by the identification of certain known spectrin defects located well outside the Nalpha 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 (Delta Galpha -lateral and Delta 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 beta -spectrin remained constant (~0.2 µM) over the range 7 mM to 1 M. The helical bundling in the Nalpha /Cbeta region has been hypothesized to be largely due to hydrophobic effects, since the interior of these three alpha -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 alpha -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 alpha -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 alpha - and beta -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 Spalpha -(1-368) (IC50 = 0.24 µM at 4 °C) was very similar to that of proteolytically digested alpha 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 alpha I fragment (Kd increasing to ~3.8 µM at 30 °C) (30), whereas our peptide exhibited a strong temperature dependence (IC50 of Spalpha -(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 alpha -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 alpha -peptide/beta -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 Nalpha /Cbeta 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 alpha beta 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 2alpha beta [dharrow] (alpha beta )2 reaction should be independent of helical bundling (Delta 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 alpha -spectrin the region consisting of residues 12-51 was involved in binding with beta -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 alpha -peptide/beta -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 Nalpha -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.

    ACKNOWLEDGEMENTS

The cDNA clone, alpha 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.

    FOOTNOTES

* 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.

Dagger 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: Nalpha region, Nalpha amino-terminal region of alpha -spectrin; Cbeta region, Cbeta carboxyl-terminal region of beta -spectrin; Spalpha -(x-y), recombinant peptide with sequence homologous to that of human erythrocyte alpha -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.
    REFERENCES
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Abstract
Introduction
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