©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping the Human Erythrocyte -Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the -Actinin Dimer Site (*)

(Received for publication, July 28, 1995; and in revised form, December 1, 1995)

Jeanine A. Ursitti (§) Leszek Kotula (¶) Tara M. DeSilva Peter J. Curtis David W. Speicher (**)

From the Wistar Institute, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-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 beta-spectrin required for association with alpha-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4) with this earlier starting point bound to full-length alpha-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 beta1-4 showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1 alone could not associate with alpha-monomers. The first two motifs (beta1-2) represented the minimum lateral dimer assembly site with a K of about 230 nM for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha18-21. 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.


INTRODUCTION

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 alpha subunit with a 246-kDa beta 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 alpha subunit (Sahr et al., 1990) and beta 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 alpha-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 alpha and beta 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 (^1)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 alphaV and betaIV domains (Speicher et al., 1992). These tryptic domains include most or all of the repetitive segments alpha19-21 and beta1-4. Interaction of these regions is apparently the initial step of dimer assembly, which is followed by subsequent lateral association of additional alpha and beta motifs. A mutation in alpha-spectrin in this region has been identified, alpha, 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 alpha mutation will decrease incorporation of the mutated chain onto the membrane. In contrast, elliptocytosis mutations on the opposite allele from the alpha mutation enhance incorporation of alpha subunit carrying the elliptocytosis mutation onto the membrane (Garbarz, 1994; Wilmotte et al., 1993). The ability of the alpha 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 beta nucleation region recombinant peptides for proper polypeptide chain folding, dimer binding affinity, and hydrodynamic properties. These analyses show that the minimum beta 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.


MATERIALS AND METHODS

Isolation of alpha-Spectrin Monomers

Spectrin was extracted from fresh human red cells within 24 h of collection and alpha-monomers were purified as described previously (Speicher et al., 1992) using a modification of the ion exchange purification initially developed by Yoshino and Marchesi(1984).

Design and Construction of beta-Spectrin Expression Plasmids

Oligonucleotide primers were designed to amplify specific regions of the beta-spectrin nucleation site from the cDNA by the polymerase chain reaction using Vent polymerase (New England Biolabs). Primers contained restriction enzyme sites for BamHI and EcoRI, at the 5` and 3` ends, respectively, to allow directional cloning of the insert into the pGEX-2T expression vector (Pharmacia Biotech Inc.).

Five beta-spectrin nucleation site clones and one alpha-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 beta motifs, beta1-4, 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 beta1-4 peptide led to the production of another clone, beta1-4, 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 beta1-4 recombinant were prepared as described in Table 1.



The entire alpha-spectrin nucleation site, encompassing repetitive motifs alpha18-21, 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 alpha recombinant, alpha1 (residues 50-158), was prepared as described previously (Kotula et al., 1993).

All expression plasmids were transformed into the DH5alpha strain of Escherichia coli. Each construct was completely sequenced to verify the integrity of the recombinant vectors. The alpha- and beta-spectrin cDNA clones were kindly provided by Dr. Bernard Forget (Yale University, New Haven, CT).

Expression and Purification of Recombinant Peptides

Overnight cultures of the cells were diluted 1:20 in LB medium containing 50 µg/ml ampicillin. Cells were grown to an optical density of 0.5-0.7 at 550 nm before induction with 1-thio-beta-D-galactopyranoside at a final concentration of 1 mM. Cells were induced for 3 h before collection by centrifugation. Cell pellets were stored at -80 °C and thawed on ice just before use. Fusion proteins were purified as described previously (Kennedy et al., 1991) with minor modifications. Briefly, each 600-ml cell pellet was resuspended in 15 ml of lysis buffer (50 mM Tris, 50 mM NaCl, 5 mM EDTA, 1 mM diisopropyl fluorophosphate, 0.15 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1% Triton X-100, pH 8.0) and sonicated to lyse the cells. The supernatant of the lysed cells was collected by centrifugation. The beta1-2 fusion protein was released into the supernatant after lysis and this supernatant could be loaded directly onto a reduced glutathione-Sepharose 4B column (Pharmacia). The fusion proteins of alpha18-21, beta1-3, beta1-4, beta1-4, and beta2-4 were primarily in inclusion bodies and were extracted in 25 ml of urea buffer (5 M urea, 50 mM Tris, 5 mM beta-mercaptoethanol, 5 mM EDTA, 1 mM diisopropyl fluorophosphate, 0.15 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, pH 8.0) using a Dounce homogenizer and incubation on ice for 2 h. The supernatant was collected and dialyzed against 500 ml of renaturation buffer (126.6 mM NaCl, 10 mM sodium phosphate, 5 mM beta-mercaptoethanol, 5 mM EDTA, 0.5 mM diisopropyl fluorophosphate, 0.15 mM PMSF, 0.1 µg/ml leupeptin, 0.1 µg/ml pepstatin, 20% glycerol, 1% Triton X-100, pH 7.3) for 2-3 h followed by an overnight dialysis against 2 liters of PBST (126.6 mM NaCl, 10 mM sodium phosphate, 5 mM EDTA, 0.15 mM PMSF, 1% Triton X-100, pH 7.3). The renatured fusion proteins were then purified on a glutathione-Sepharose 4B column. Peptides were cleaved from the GST molecule in the elution buffer (50 mM Tris, 10 mM reduced glutathione, pH 8.0) using bovine thrombin (Sigma). NaCl (final concentration 150 mM) was added to the elution buffer prior to thrombin digestion for the beta1-4 and beta1-3 fusion products to decrease the formation of secondary cleavage products. The ratio of thrombin to fusion protein and the potential advantage of raising the ionic strength was determined empirically for each fusion protein. The optimal thrombin cleavage conditions were: beta1-4, 20 units/mg; beta1-4, 10 units/mg; beta1-3, 2 units/mg; beta1-2, 4 units/mg; beta2-4, 1 unit/mg; and alpha18-21, 1 unit/mg for 3 h at 37 °C. Cleaved peptides were purified by rechromatography on a glutathione-Sepharose column followed by HPLC gel filtration on two preparative (21.5 times 600 mm) TSK-gel columns (G3000SW + G2000SW) in series (TosoHaas) in phosphate-buffered saline (126.6 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, 0.15 mM PMSF, 0.05% sodium azide, pH 7.3).

Analytical HPLC Gel Filtration Binding Assay

Spectrin alpha-monomers or the alpha18-21 peptides were mixed with purified recombinant nucleation site beta-peptides and incubated at 0 °C for different times ranging from 5 min to 15 h. Under most conditions equilibrium was reached within 5 to 15 min, hence a 25-min incubation time was used for most binding assays. To determine Stokes' radius, the proteins and peptides were separated on two analytical (7.8 times 300 mm) TSK-gel columns (G3000SW + G2000SW) at 4 °C with a flow rate of either 0.4 or 0.8 ml/min. Binding experiments used only the G3000SW column at 1.0 ml/min at 4 °C in phosphate-buffered saline buffer. Eluted proteins were detected by absorbance at 280 nm and intrinsic tryptophan fluorescence (excitation 280 nm, emission filter 370 nm) and were quantified on a data acquisition system (PE Nelson Analytical) using peak area. Response factors for each protein were determined by replicate injections of known quantities (determined by quantitative amino acid analysis) for each component. Molecular weights used for calculating molarity were: alpha-monomer, 280,000; beta1-4, 52,085; beta1-4, 52,964; beta1-3, 40,374; beta1-2, 27,998; beta1, 15,681; beta2-4, 38,924; alpha18-21, 51,938. Association (K(a)) and dissociation constants (K(d)) for binding of beta-spectrin nucleation region peptides with intact alpha-spectrin monomers were determined by calculating the amount of unbound peptide relative to control samples under the same conditions.

Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was performed using 7 and 10% slab gels (1.5 times 100 mm) as described by Laemmli(1970). For low molecular weight components, 5-15% gradient Tricine gels were used (Schagger and von Jagow, 1987). All gels were stained with Coomassie Brilliant Blue R-250.

N-terminal Sequence Analysis

After separation by SDS-polyacrylamide gel electrophoresis, peptides were transferred onto high retention polyvinylidene difluoride membranes (Bio-Rad) as described previously (Mozdzanowski et al., 1992). After staining with Amido Black, the bands of interest were excised and sequenced on a Hewlett-Packard G1005A sequencer as described previously (Reim and Speicher, 1994).

Circular Dichroism (CD) Measurements

CD spectra were performed on a Jasco J720 instrument at room temperature in a 0.2-mm path length cell. Proteins were in phosphate-buffered saline buffer, pH 7.3, and protein concentrations were determined by duplicate quantitative amino acid analysis.

Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was performed on a PerSeptive Biosystems Vestec Mass Spectrometer using Voyager software. Proteins were dialyzed into 20 mM ammonium bicarbonate, pH 8.0, 1 µl of sample was mixed with 1 µl of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid for samples <20 kDa and sinapinic acid for samples >20 kDa in 0.1% trifluoroacetic acid, 33% acetonitrile), the sample/matrix mixture was transferred to the sample target, dried, and analyzed. Expected masses were calculated from known sequences using the GPMAW program (Lighthouse Data, Denmark).


RESULTS

Design and Characterization of beta-Spectrin Nucleation Site Recombinant Peptides

As noted above, previous results using spectrin peptides from mild protease cleavage of purified spectrin monomer mapped the spectrin dimer nucleation site to approximately the last three to four homologous motifs of the alpha subunit and the first four homologous motifs of the beta subunit (Speicher et al., 1992). To determine the minimal nucleation site requirements of beta-spectrin as well as physical properties of this region, a series of recombinant peptides were produced by truncating the previously defined beta-spectrin nucleation region (beta1-4) at either the N terminus or the C terminus. In addition, an alpha-spectrin nucleation site peptide was constructed that included the entire putative nucleation site region on the alpha subunit, alpha18-21. The sequence content of each peptide is shown diagrammatically in Fig. 1A and their relationship to the overall motif structure of a spectrin dimer is shown in Fig. 1C. As illustrated, the phasing of each recombinant peptide correlates with the boundaries of homologous triple helical motifs as defined by the high resolution structure of a single motif determined by Yan et al.(1993) with the exception of the ``+'' series of beta-peptides, which begin 8 residues before the predicted repetitive segment (see below).


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 beta peptides. This series of peptides begins 8 residues earlier than the normal conformational motif. The C-terminal end of the beta1 (*), beta1-2 (), and beta1-3 () peptides are marked as indicated. B, SDS gels of alpha-monomers and recombinant peptides after cleavage from the GST fusion moiety and repurification. The samples are: lanes 1 and 2 (4 µg/lane), alpha-spectrin monomer and alpha18-21, respectively, on a 7% Laemmli gel; lanes 3-7 (2 µg/lane), beta1-4, beta1-4, beta1-3, beta2-4, and beta1-2, respectively, on a 10% Laemmli gel; and lane 8 (2 µg/lane), beta1 on a 5-15% linear gradient Tricine gel. C, arrangement of the structural motifs in an anti-parallel spectrin dimer. The beta 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 alpha 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 beta1 peptide was isolated as a proteolytic by-product of the beta1-2 construct. During purification of the beta1-2 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 beta1 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 beta1-2 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 beta1-4 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 beta1. The full-length beta1-4 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.

Proper Phasing Results in a Less Constrained Conformation of the beta1-4 Peptide

The unusual difficulty encountered with thrombin cleavage of the GST-beta1-4 construct relative to other GST fusion proteins containing complete spectrin motifs (see below) led to further evaluation of the beta1 start point. To examine the relationship between the longer alpha-actinin and spectrin nucleation region motifs relative to the more common 106-residue motif, the last four alpha-spectrin (alpha18-21), the first four beta-spectrin (beta1-4), and the four spectrin-type motifs of human cytoskeletal alpha-actinin were aligned against numerous 106-residue spectrin motif sequences using the computer program ALIGN to perform optimized pairwise alignments. Gaps and insertions of the longer motifs relative to the 106-residue motif and its associated crystallographic structure were placed (Fig. 2A) in the most frequently aligned position from the individual pairwise comparisons. Two observations emerged from this alignment. First, all insertions larger than a single residue mapped to two regions, an 8-residue segment near the beginning of the motif that probably extends the A helix as previously suggested (Viel and Branton, 1994), and variable length insertions that map to the turn region between helices B and C. Second, a number of prolines are located in positions that were helical in the reported high resolution structure of the Drosophila alpha14 motif (Yan et al., 1993), which supports the hypothesis that the nucleation site motifs have a unique conformation responsible for initiating dimerization.


Figure 2: Correlation of the elongated spectrin and alpha-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 (alpha18-21 and beta1-4) and the spectrin-like motifs of alpha-actinin that are responsible for alpha-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 alpha-actinin produced by limited proteolysis. Mild cleavage of alpha-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 beta-spectrin with trypsin removes the ABD domain by cleavage at residue 292 and the earliest observed fragments that contain the beta 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 beta 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 alpha-actinin motifs (A2, A3, and A4) and their most homologous spectrin counterparts (beta2, alpha20, and alpha21, 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 beta1-4 recombinant compares favorably with the observed mild protease cleavage sites for both alpha-actinin and beta-spectrin as shown in Fig. 2B. In addition, although various start sites have been reported for the alpha-actinin motif based on alignments of sequences, the revised start site presented here for the first motif of spectrin and alpha-actinin agrees with the recent phasing analysis of alpha-actinin using recombinant peptides reported by Gilmore et al.(1994).

To further evaluate the phasing of the first beta motif, a beta1-4 recombinant peptide, which started at residue 293 compared with residue 301 for the beta1-4 peptide, was produced and analyzed in parallel with the beta1-4 peptide. Thrombin cleavage of the beta1-4 and beta1-4 fusion proteins differed markedly at physiological ionic strength. The beta1-4 fusion protein was efficiently cleaved without production of secondary cleavage products. In contrast, under the same conditions, <50% of the beta1-4 protein was cleaved and a prominent secondary cleavage product was formed (data not shown).

Secondary Structure and Hydrodynamic Properties of beta-Spectrin Recombinant Peptides

All spectrin recombinant peptides were analyzed by circular dichroism to determine whether the peptides were properly folded. Representative spectra are shown in Fig. 3and all peptides reported here had high alpha-helicity (about 80%) similar to spectrin dimers and monomers.


Figure 3: CD spectra of alpha-monomer and representative recombinant peptides. bullet-bullet-, alpha-monomer (0.47 mg/ml); -, beta1-4 (0.33 mg/ml); - - - -, beta1-3 (0.47 mg/ml); and - -, beta1-2 (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^2/dmol.



The Stokes' radii of the beta-spectrin nucleation site recombinant peptides and a single motif alpha subunit peptide, alpha1 (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 beta1 or alpha1, 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 beta1-4 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 beta1-4 peptide, however, it did have normal helicity as determined by circular dichroism.


Figure 4: Hydrodynamic properties of recombinant beta-spectrin nucleation site peptides. The Stokes' radii of beta-spectrin recombinant peptides were determined by HPLC gel filtration. Peptides include beta1-4 (), beta1-4 (+), beta1-3 (+), beta2-4 (box), beta1-2 (+), beta1 (+), and alpha1 (circle) with duplicate determinations shown for beta1-4. The alpha1 motif peptide (residues 50-158) is described in Kotula et al.(1993). The more compact beta1-4 peptide was not included in the linear regression plot. Slope = 0.85, y intercept = 1.5, R = 0.9994.



Functional Analysis of Recombinant Peptides from the beta-Spectrin Dimer Nucleation Site Region

In order to identify the minimal requirements for the beta-spectrin nucleation site and the effects of additional motifs on binding affinity, the recombinant beta peptides were evaluated in solution binding assays with either purified native alpha-spectrin monomers or recombinant alpha18-21. Time course experiments showed that binding equilibrium was usually reached within 5 min or less under most concentrations and molar ratios evaluated. Therefore, protein mixtures were routinely incubated for 25 min prior to measurement of complex formation using a rapid HPLC gel filtration separation as shown in Fig. 5. Free alpha-monomers could not be resolved from complexes due to the small change in size when the complex was formed. Therefore, carefully quantified amounts of alpha-monomers and recombinant peptides were combined and association constants were determined by measuring the loss of recombinant peptide from its normally eluting position relative to an identical control without alpha-monomer. Control experiments showed that only equimolar binding occurred and that any dissociation of complex that occurred during the analysis did not increase the area of the unbound recombinant peptide peak. As shown in Fig. 5, no detectable binding was observed for the beta1 or beta2-4 peptides. In addition, no binding to alpha-monomers was detected for these two proteins when as much as a 3- or 5-fold molar excess of recombinant peptide was used. The beta1 peptide, although required for nucleation site binding, is apparently not sufficient.


Figure 5: Binding of beta-spectrin recombinant peptides to alpha-monomers. Purified recombinant peptides were mixed with alpha-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 alpha-monomer. A, solid lines are chromatograms of association experiments where 500 pmol of the recombinant beta peptide indicated in the panel was mixed with 500 pmol of alpha-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 (alpha-monomer portion of gel not shown).



Binding affinities of the recombinant beta peptides with alpha-monomers are summarized in Table 2. The importance of N-terminal phasing for the first beta motif is illustrated by the observation that the beta1-4 peptide has nearly a 10-fold lower affinity for alpha-monomers compared with the 8-residue longer beta1-4 peptide. The minimum dimerization site contains the first two motifs, which has a K(d) 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(d) of about 10 nM.



The lateral association of beta recombinant peptides with alpha-monomers is readily reversible. As shown in Fig. 6, both the high affinity beta1-4 peptide and the lower affinity beta1-2 peptides can compete with each other for binding to alpha-spectrin monomers. beta1-2 was also able to compete with beta1-3 for binding to alpha-spectrin (data not shown).


Figure 6: Competition of beta-spectrin nucleation site peptides for association with alpha-monomers. Dashed lines are control experiments where alpha-monomers and a beta 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 beta nucleation site peptide and were incubated for an additional 25 min prior to HPLC separation. See Fig. 5A for additional details. Upper panel: - - - -, alpha-monomer + beta1-4; -, alpha-monomer + beta1-4 + 5 times beta1-2. Lower panel, - - - -, alpha-spectrin + beta1-2, -, alpha-spectrin + beta1-2 + 5 times beta1-4.



The Actin Binding Domain Is Not Required for Dimer Assembly and Does Not Substantially Contribute to Dimer Affinity

As illustrated above, high affinity dimers can readily form without the presence of the N-terminal actin binding domain. To further evaluate whether the actin binding domain may contribute positively or negatively to dimer assembly, binding measurements of intact beta subunits to the alpha18-21 recombinant peptide were performed. The K(d) for this interaction between an intact beta subunit and a 4-motif alpha peptide is about 15 nM which compares favorably with the K(d) of 10 nM observed for a 4-motif beta peptide interaction with intact alpha-monomers.

Noncovalent Associations between Helices within a Single Motif Are High Affinity Interactions That Frequently Maintain Functional Complexes

As described above, the dramatic reduction in affinity of the beta1-4 peptide (8-residue shorter N-terminal) compared with beta1-4 suggested that additional truncation of the beta1 motif would further reduce or abolish binding. In this context, an apparently inconsistent observation was that the 43-kDa peptide, the secondary cleavage product of the beta1-4 recombinant, did show detectable binding to alpha-monomers. In the experiment shown in Fig. 5B (lane 3), a faint 43-kDa band was observed on the original gel and some preparations of the beta1-4 peptide showed even more extensive binding of the 43-kDa band to alpha-monomers than the illustrated experiment. As noted above, N-terminal sequence analysis of this peptide showed that it was cleaved at residue 374 and lacked helices A and B of the beta1 motif. It was therefore quite surprising that this truncated form of the molecule could effectively compete with a larger amount of intact beta1-4, which was always present in these preparations, for association with alpha-monomers.

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.


DISCUSSION

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 beta 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 beta-spectrin nucleation site represent functionally important findings.

The minimum beta peptide capable of dimerizing to the alpha subunit contains the first two homologous motifs (beta1 and beta2), and each additional motif (beta3 and beta4) increases the binding affinity approximately 5-fold, apparently by forming additional lower affinity lateral associations with a complementary motif in the alpha-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-beta1-4 or GST-alpha18-21) is used instead of the complementary monomer (Speicher et al.(1992) and data not shown).

The beta1 motif is required for dimerization, but is insufficient for high affinity association as shown by the loss of dimer formation capacity of the native beta2-4 and the beta1 recombinant peptides (Fig. 5). In addition, these experiments showed that the precise phasing (starting point) of the beta1 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 beta1 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 alpha20, alpha21, and beta2 (Sahr et al., 1990; Winkelmann et al., 1990) and near the beginning of Drosophila spectrin motifs alpha20, alpha21, and beta2 (Viel and Branton, 1994). The corresponding start site for the closely related first alpha-actinin motif had not been clearly defined, with reported possibilities ranging from alpha-actinin residue 245 to 266 (Baron et al., 1987; Imamura et al., 1988; Blanchard et al., 1989). The difficulty encountered in cleaving a beta1-4 fusion protein using the phasing defined for 106-residue spectrin-type motifs and further consideration of the locations for mild protease cleavage sites for beta-spectrin and alpha-actinin (Fig. 2B) suggested that both the first beta motif and the first alpha-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 beta1-4 peptide has an order of magnitude lower binding affinity for alpha-monomers compared with the beta1-4 peptide and its affinity is even lower than the beta1-3 peptide. The motif phasing determined experimentally for the beta1 motif and inferred for the first alpha-actinin motif in this study is consistent with the alpha-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 beta 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 beta-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 alpha21 motif (EF-hand motifs) and before the beta1 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 beta1-4 recombinant could associate with either alpha-spectrin monomers or an alpha18-21 recombinant protein with a K(d) of approximately 10 nM and intact beta-monomers bind to the alpha18-21 recombinant with a similar affinity. Although these experiments do not rule out a direct interaction of the alpha EF-hand motifs with the beta-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 alpha subunit (Speicher et al.(1992) and data not shown). It is particularly surprising that the Drosophila beta1-4Delta288 peptide is inactive since it corresponds closely to our beta1-3 recombinant, which has a K(d) 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 beta-fodrin that contain only a portion of the actin binding domain. Since the present study shows that the phasing of the beta1 motif has a dramatic effect on dimer binding affinity, it is most likely that some Drosophila beta 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 beta1-4 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 alpha (DiPaolo et al., 1993) and beta 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 alpha and beta 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 alpha1 peptide and the nucleation site beta1 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL38794 and National Science Foundation Grant MCB 9304746 (to D. W. S.), National Institutes of Health Grants HL08840 (to J. A. U.) and HL33884 (to P. J. C.), and by partial support from National Cancer Institute Cancer Core Grant CA10815. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: University of Maryland School of Medicine, Dept. of Physiology, 655 W. Baltimore St., Baltimore, MD 21201.

Present address: Laboratory of Molecular Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314.

**
To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3972; Fax: 215-898-0664.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; PMSF, phenylmethylsulfonyl fluoride: GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI, matrix-assisted laser desorption/ionization.


ACKNOWLEDGEMENTS

We thank Dr. Laszlo Otvos, Jr. (The Wistar Institute) for performing the CD measurements and for assistance in interpreting this data. We also thank Dr. Bernard Forget (Yale University, New Haven, CT) for providing spectrin cDNA clones.


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