Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side ...

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THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc Vol. 267, No. 21, Issue of July 25, pp. 14775-14782,1992 Printed in U. S. A Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side) Assembly and Identificationof an Essential Nucleation Site* (Received for Publication, January 29, 1992) David W. SpeicherS, Ludmila Weglarz, and Tara M. DeSilva From the Wistar Institute for Anatomy and Biology, Philadelphia, Pennsylvania 19104 The antiparallel side-to-side association of spectrin a and j3 monomers is a two-step process which occurs in seconds even at 0 “C and atlow concentrations. Assembly involvesinitial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homolo- gous segments or repeats. Three repeats in the nuclea- tion site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in a-actinin. The adjacent actin binding domain on the ,B subunit and the adjacent EF hand motifs on the a subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both sub- units. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associ- ate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Het- erozygotic mutations involving either nucleation site are predicted to affectallele incorporation into the mature membrane skeleton. Spectrin is a major, central component of a largely two- dimensional protein network on the cytoplasmic face of the red cell membrane. This membrane skeleton is primarily responsible for the distinctive biconcave shape as well as the unique structural integrity and deformability of the red cell. Closely related spectrin isoforms, usually associated with membranes, are now known to occur in most other tissues and even in single cell organisms. The flexible and dynamic red cell membrane skeleton is comprised of spectrin tetramers, short actin oligomers, Protein 4.1, ankyrin, andseveral other proteins (for recent reviews see Bennett and Lambert, 1991; Morrow, 1989). The critical role of spectrin in maintaining membrane properties is clearly demonstrated by hereditary hemolytic anemias which involve quantitative spectrin defi- ciencies and/or spectrin structural mutations (Palek, 1987; McGuire and Agre, 1988; Marchesi, 1989). * This work was supported by National Institutes of Health Grant HL38794 and a 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 be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondence andreprintrequestsshould be ad- dressed The Wistar Institute, 3601 Spruce St., Room C102, Phila- delphia, PA 19104-4268. Tel.: 215-898-3972. Fax: 215-898-0664. Most of the elongated, flexible spectrin molecule is com- prised of a series of contiguous, homologous motifs approxi- mately 106 residues in length (Speicher and Marchesi, 1984; Speicher, 1985). The complete sequences of the human red cell 280-kDa a subunit (Sahr et al., 1990) and the 246-kDa (3 subunit (Winkelmann et al., 1990), deduced from cDNA se- quences, confirm that most of the molecule is comprised of these repetitive segment motifs or repeats. Additional struc- tural features deduced from the complete sequence include an actin binding domainat the N-terminal of the (3 subunit and a pair of EF hand motifs at the C-terminal of the a subunit. Red cell spectrin is the prototypicalmember of a protein superfamily which includes fodrin (non-red cell spectrins), a- actinins (Wasenius et d., 1987; Baron et d., 1987), and dystrophins (Davison and Critchley, 1988; Koenig et al., 1988). The spectrin repeat motif is the predominant structural ele- ment in these other family members although the average length of the repeat is generally slightly longer and less precisely preserved in a-actinin and dystrophin. The actin binding domainmotif and EF hand motifs are also present in a-actinin and dystrophin. Although the structure and dynamics of protein-protein interactionsinthe red cell membraneskeleton have been extensively studied for many years, relatively little is known about the initial assembly step to form spectrin dimers. Spec- trin a and @ subunits associate side-to-side in an antiparallel orientation (Speicher et al., 1982) to produce heterodimers that are approximately the same contour length as the isolated monomers (Yoshino and Marchesi, 1984; Coleman et al., 1989). This strong subunit-subunit interaction can only be dissociated by strong chaotrophic reagents or detergents such as SDS.’ The mildest method for dissociation employs ion exchange chromatography in the presence of 3 M urea (Yosh- ino and Marchesi, 1984). Yoshino and Marchesi showed that reconstituted heterodimers purified by this methodrecovered native tryptophan fluorescence anisotropy, migrated normally on native gels, and resembled native dimers by electron mi- croscopy. The purified @ subunit hada morphology similar to dimers and migrated as a discrete band on native gels, while the a subunit was heterogeneous by both analysis methods. Although apparently native heterodimer reconstitution could be achieved, the kinetics and mechanism of dimer assembly has not been defined. Detailed characterization of heterodi- mer assembly has been limited by the relatively slow (24-48 h) native gel electrophoresis method used to separate and quantify spectrin dimers and monomers in most previous studies. Several structural features of a.(3 side-to-side association, which probably involves primarily the repetitive motifs (Im- The abbreviations used are: SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophosphate; HPLC, high performance liquid chro- matography; PMSF, phenylmethylsulfonylfluoride; PVDF, polyvi- nylidene difluoride. 14775

Transcript of Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side ...

Page 1: Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 21, Issue of July 25, p p . 14775-14782,1992 Printed in U. S. A

Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side) Assembly and Identification of an Essential Nucleation Site*

(Received for Publication, January 29, 1992)

David W. SpeicherS, Ludmila Weglarz, and Tara M. DeSilva From the Wistar Institute for Anatomy and Biology, Philadelphia, Pennsylvania 19104

The antiparallel side-to-side association of spectrin a and j3 monomers is a two-step process which occurs in seconds even at 0 “C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homolo- gous segments or repeats. Three repeats in the nuclea- tion site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in a-actinin. The adjacent actin binding domain on the ,B subunit and the adjacent EF hand motifs on the a subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both sub- units. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associ- ate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Het- erozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.

Spectrin is a major, central component of a largely two- dimensional protein network on the cytoplasmic face of the red cell membrane. This membrane skeleton is primarily responsible for the distinctive biconcave shape as well as the unique structural integrity and deformability of the red cell. Closely related spectrin isoforms, usually associated with membranes, are now known to occur in most other tissues and even in single cell organisms. The flexible and dynamic red cell membrane skeleton is comprised of spectrin tetramers, short actin oligomers, Protein 4.1, ankyrin, and several other proteins (for recent reviews see Bennett and Lambert, 1991; Morrow, 1989). The critical role of spectrin in maintaining membrane properties is clearly demonstrated by hereditary hemolytic anemias which involve quantitative spectrin defi- ciencies and/or spectrin structural mutations (Palek, 1987; McGuire and Agre, 1988; Marchesi, 1989).

* This work was supported by National Institutes of Health Grant HL38794 and a 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 be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely t o indicate this fact.

$ T o whom correspondence and reprint requests should be ad- dressed The Wistar Institute, 3601 Spruce St., Room C102, Phila- delphia, PA 19104-4268. Tel.: 215-898-3972. Fax: 215-898-0664.

Most of the elongated, flexible spectrin molecule is com- prised of a series of contiguous, homologous motifs approxi- mately 106 residues in length (Speicher and Marchesi, 1984; Speicher, 1985). The complete sequences of the human red cell 280-kDa a subunit (Sahr et al., 1990) and the 246-kDa (3 subunit (Winkelmann et al., 1990), deduced from cDNA se- quences, confirm that most of the molecule is comprised of these repetitive segment motifs or repeats. Additional struc- tural features deduced from the complete sequence include an actin binding domain at the N-terminal of the (3 subunit and a pair of EF hand motifs at the C-terminal of the a subunit. Red cell spectrin is the prototypical member of a protein superfamily which includes fodrin (non-red cell spectrins), a- actinins (Wasenius et d., 1987; Baron et d . , 1987), and dystrophins (Davison and Critchley, 1988; Koenig et al., 1988). The spectrin repeat motif is the predominant structural ele- ment in these other family members although the average length of the repeat is generally slightly longer and less precisely preserved in a-actinin and dystrophin. The actin binding domain motif and EF hand motifs are also present in a-actinin and dystrophin.

Although the structure and dynamics of protein-protein interactions in the red cell membrane skeleton have been extensively studied for many years, relatively little is known about the initial assembly step to form spectrin dimers. Spec- trin a and @ subunits associate side-to-side in an antiparallel orientation (Speicher et al., 1982) to produce heterodimers that are approximately the same contour length as the isolated monomers (Yoshino and Marchesi, 1984; Coleman et al., 1989). This strong subunit-subunit interaction can only be dissociated by strong chaotrophic reagents or detergents such as SDS.’ The mildest method for dissociation employs ion exchange chromatography in the presence of 3 M urea (Yosh- ino and Marchesi, 1984). Yoshino and Marchesi showed that reconstituted heterodimers purified by this method recovered native tryptophan fluorescence anisotropy, migrated normally on native gels, and resembled native dimers by electron mi- croscopy. The purified @ subunit had a morphology similar to dimers and migrated as a discrete band on native gels, while the a subunit was heterogeneous by both analysis methods. Although apparently native heterodimer reconstitution could be achieved, the kinetics and mechanism of dimer assembly has not been defined. Detailed characterization of heterodi- mer assembly has been limited by the relatively slow (24-48 h) native gel electrophoresis method used to separate and quantify spectrin dimers and monomers in most previous studies.

Several structural features of a.(3 side-to-side association, which probably involves primarily the repetitive motifs (Im-

The abbreviations used are: SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophosphate; HPLC, high performance liquid chro- matography; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvi- nylidene difluoride.

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14776 Nucleation Site for Spectrin Dimer Assembly

amura et al., 1988), remain unresolved. The register of side- to-side pairing of the shorter p subunit (17 repeats) with the longer a subunit (20 to 21 repeats) and the mechanism(s) that limit this pairing to a single unique register have not been determined. The number of side-to-side interaction sites is also ambiguous. Rotary-shadowed images of native spectrin dimers show strong side-to-side interactions only at the phys- ical ends of the dimers (Shotton et al., 1979), while peptide mapping indicated multiple points of attachment along the two subunits (Morrow et al., 1980). Morphometric analysis of rotary-shadowed images of spectrin monomers and reconsti- tuted dimers suggested that the overall flexibility and linearity of dimers and tetramers is contributed by the p subunit (Coleman et al., 1989). This study determined that no addi- tional rigidity was contributed by noncovalent side-to-side a . ,6 associations suggesting limited side-to-side interaction. Fur- ther characterization of this first, critical step of spectrin assembly is also of interest since heterodimer assembly mu- tations have recently been identified (Alloisio et al., 1991).

In this study, a rapid HPLC gel filtration assay is used to study heterodimer reassembly as well as the flexibility of isolated monomers and reconstituted heterodimers. Peptide- monomer reassembly experiments show that only selected peptides which contain an essential nucleation site near the actin binding domain can reassociate side-to-side, and this site determines the side-to-side register or pairing of a and 0 repeat segments.

EXPERIMENTAL PROCEDURES

Extraction of Spectrin-Human red cells were obtained from healthy donor blood as previously described with minor modifications (Litman et al., 1980). Briefly, a unit of whole blood was processed at a time and within several hours of collection. All steps were carried out at 0 "C to 4 "C except as noted. Serum and buffy coat were removed after centrifugation at 4500 X g for 15 min, and the red cells were washed three times in isotonic phosphate-buffered saline. Washed red cells were typically stored overnight in phosphate-buff- ered saline containing 10 mM glucose and 0.1 mM PMSF. The cells were lysed in a total volume of 2.5 liters by the addition of cold lysing buffer (5 mM sodium phosphate, 1 mM EDTA, 0.04 mM DFP (Sigma), pH 8.0) for 30 min. Hemoglobin-free membranes were prepared by repeated washes (typically five) with lysing buffer and collection of the membranes by centrifugation at 30,000 X g for 35 min. Spectrin was extracted by incubating the washed membranes at 37 "C for 20 min in 5 to 10 volumes of extraction buffer (0.1 mM EDTA, 0.5 mM 2-mercaptoethanol, 0.2 mM DFP, pH 9.0). The vesiculated ghosts were separated from the extracted spectrin by centrifugation at 47,900 X g for 60 min. The supernatant, primarily spectrin and actin, was referred to as crude spectrin and stored on ice.

Isolation of Spectrin Monomers-Spectrin monomers were isolated essentially as described by Yoshino and Marchesi (1984) with the following modifications. Crude spectrin was used instead of purified spectrin dimer as the initial sample. Crude spectrin extracts were adjusted to 3 M urea, 150 mM NaCl, 20 mM Tris, 1 mM EDTA, and 0.6 mM 2-mercaptoethanol by the addition of solid urea and 200 mM Tris-HC1 buffer, pH 8.2, containing 1.5 M NaCl and 10 mM EDTA. This sample was then mixed batchwise with DEAE-cellulose (What- man, DE52), pre-equilibrated with 18 mM Tris-HC1 buffer, pH 8.2, containing 3 M urea, 135 mM NaCI, and 0.9 mM EDTA, instead of applying the sample to a DEAE-cellulose column. After incubation for 1 h at 4 "C, the sample/DEAE slurry was packed into a 5-cm inside diameter column (300 ml of resin per 100 mg of crude spectrin) and washed with equilibration buffer. Proteins were eluted at 4 ml/ min using equilibration buffer containing increasing concentrations of NaCl in the four steps indicated below. Actin and other weakly bound contaminants were eluted with buffer containing 152 mM NaCl. The p monomers of spectrin were eluted by 181 mM NaCl after which the concentration was changed to 234 mM NaCl to elute a monomers. The fractions were analyzed on slab gels containing 0.1% SDS by the method of Laemmli (1970). Pooled fractions containing separated monomers were concentrated using vacuum dialysis in a Micro-ProDiCon concentrator against isotonic KC1 buffer (10 mM Tris, 20 mM NaC1, 130 mM KCl, 1 mM 2-mercaptoethanol, 30 p M

PMSF,pH 7.4). HPLC Purification of Spectrin Monomers-For most experiments,

concentrated spectrin monomers were further purified by HPLC gel filtration to remove trace levels of residual dimers and improperly folded monomers using either three analytical TSK 5000 PWXL columns (7.8 mm X 30 cm) in series or one preparative TSK 5000 PW column (21.5 mm X 60 cm) in isotonic KC1 buffer, pH 7.4. Typically, a flow rate of 0.3 ml/min was used for the 7.8-mm column, and 1.0 ml/min was used for the 21.5-mm column. Pooled monomer peak fractions were concentrated under vacuum and further dialyzed against two changes of isotonic KC1 buffer.

Mild Proteolytic Digestion of Spectrin Monomers-Purified spec- trin monomers were dialyzed into isotonic KC1 buffer without PMSF, pH 8.0, immediately before protease cleavage, and the concentration of monomers was adjusted to 1 mg/ml or less. Trypsin (L-l-tosylam- ido-2-phenylethyl chloromethyl ketone-treated, Worthington) was used for all digestions at 0 "C. In several experiments, aliquots of spectrin monomers were digested in 20 mM Tris, pH 8.0, containing 0.02% sodium azide, and 1 mM 2-mercaptoethanol (TAB buffer) instead of isotonic KC1 buffer. A wide range of intermediate-sized peptides of each monomer were produced using enzyme-to-substrate ratios ( E / S ) of 1:20, 1:100, or 1:800 and cleavage times of either 90 min or 6 h. Enzymatic hydrolysis was terminated by the addition of DFP to a final concentration of 1 mM. The extent of digestion of the spectrin monomers was monitored on one-dimensional 7% or 13% Laemmli gels.

Reassociation of Monomer Peptides with Intact Complementary Subunit-Reassembly of monomer peptides to the complementary subunit was examined by combining unfractionated monomer peptide mixtures with complementary intact subunit for a range of times (0 min to 24 h) followed by HPLC gel filtration using three analytical TSK 5000 PWXL columns (7.8 mm X 30 cm) in series in isotonic KC1 buffer, pH 7.4. The flow rate for analytical assays was 0.4 ml/ min. The injection loop and columns were maintained at 2-4 "C in most experiments. Components were detected by absorbance at 280 nm and by intrinsic tryptophan fluorescence (excitation 280 nm, emission filter 370 nm). Chromatographic fractions were analyzed on SDS-polyacrylamide gels. In some experiments, fractions containing either monomer with bound peptides or unbound peptides were separately pooled and dialyzed against TAB buffer, pH 7.6, lyophi- lized, and electroblotted from SDS gels for N-terminal peptide se- quence analysis and for immunoblot analysis.

Western Blotting-Spectrin monomer peptides separated on one- dimensional SDS gels were blotted onto PVDF membranes (Immo- bilon P, Millipore) essentially as previously described (Mozdzanowski et al., 1992). Briefly, electrotransfer was carried out in 96 mM glycine, 12.5 mM Tris, pH 8.3, containing 10% methanol for 3 h at 250 mA. Membranes were washed three times for 15 min in Milli-Q water, then three times for 5 min in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 8.0). The rinsed PVDF membranes were blocked overnight at 4 "C in TBST containing 5% nonfat dry milk, incubated for 1 h at room temperature with either mouse monoclonal (Yur- chenco et al., 1982) or rabbit polyclonal antibodies raised against various spectrin domains; incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat antibodies to either rabbit or mouse IgG (Promega), and finally incubated with substrate solution containing 100 mM Tris, pH 9.5, 100 mM NaC1,5 mM MgClz, 0.3 mg/ ml nitro blue tetrazolium, and 0.15 mg/ml5-bromo-4-chloro 3-indoyl phosphate. The color reaction was stopped by rinsing the immuno- blots in Milli-Q water. TBST was used for all antibody dilutions and membrane rinses.

Identification of Complexed Peptides by N-terminal Sequence Analysis-After separation by SDS-gel electrophoresis, tryptic pep- tides were transferred onto high retention PVDF membranes (either Bio-Rad or Applied Biosystems) as previously described (Mozdza- nowski and Speicher, 1990). After staining with Coomassie blue, the peptide bands were cut out, destained in 40% methanol, and se- quenced on an Applied Biosystems model 475 sequenator equipped with an on-line model 120A HPLC for phenylthiohydantoin amino acid identification. Sequence reagents and programs were optimized for high sensitivity sequence analysis on PVDF membranes as pre- viously described (Speicher, 1989; Reim et al., 1992).

RESULTS

Hydrodynamic Properties of Isolated Spectrin Monomers and Analysis of Heterodimer Assembly-Purified spectrin (Y

and 0 monomers elute from HPLC gel filtration columns as

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Nucleation Site for Spectrin Dimer Assembly 14777

0 10 20 30 40 50 60 70 EO SO

Time (min)

FIG. 1. Assembly of heterodimers from purified spectrin monomers using a rapid analytical HPLC assay. Separation of spectrin monomers and dimers by HPLC gel filtration in isotonic KC1 buffer a t 0.4 ml/min on three TSK 5000 PWXL columns (7.8 mm X 30 cm) in series a t 4 "C. Absorbance is in arbitrary units with the largest peak scaled to a uniform height in all chromatograms. The retention times of spectrin tetramers (T), dimers (D), and monomers (M) are indicated. A, 100 pg of purified spectrin at 5 mg/ ml containing dimers and tetramers; B, 100 pg of 8 monomers a t 1.25 mg/ml; C , 100 pg of a monomers at 1.84 mg/ml; D, 2 pmol of CY

monomers and 2 pmol of 8 monomers, final concentration 1.7 mg/ ml, injected within seconds of combining the monomers.

TABLE I Identification of peptide complexes after HPLC separation

After mild trypsin digestion of dimers ( E / S = 1:200,0 "C, 90 rnin), aliquots from the dimer region and later-eluting peaks were separated on a 5-15% gradient gel and blotted to a PVDF membrane. The two peptides that dissociated from the major dimer region complex (bands a and b) as well as several bands in the dimer region complex (bands c-e) were excised and sequenced as described under "Experimental Procedures" to identify the peptides and determine their position in the molecule.

Band M. Seauence Location' Domainb

a 80,000 ETVVESSGPK . . . a-6 CUI b 22,000 ALADEREVVQ.. . 8-47 DAB' c 46,000 GTQLHEANQQ.. . CY-680 a11 d 35,000 GTQLHEANQQ . . . CY-680 a11 e 20,000 VHTAFERELH.. . 8-1837 81

"Residue number in the complete a (Sahr et al., 1990) or 8 (Winkelmann et al., 1990) sequences is indicated.

Nomenclature for domains as previously described (Speicher et al., 1982; Speicher and Marchesi, 1984).

AB = actin binding site. This peptide represents the actin binding domain which was not identified in the original spectrin domain map.

discrete, symmetrical bands with a smaller Stokes radius than spectrin dimers (Fig. 1). Since their contour lengths are sim- ilar to normal dimer contour lengths, the monomers must be more flexible than dimers under these conditions in contrast to the similar flexibility of monomers and dimers observed by electron microscopy (Coleman et al., 1989). Elution positions of both LY and p monomers are identical (52.2 & 0.1 min), indicating no significant difference in hydrodynamic shape. The conditions shown in Fig. 1 include a helium sparging system on the buffer to maintain flow rate reproducibility, and, during a series of analyses, the range in retention times for the same sample is less than kO.1 min. Therefore, very small shifts in migration could be reproducibly detected by this method.

An important early observation from the HPLC analysis

A

# I I I I l I I I I

0 10 20 30 40 50 60 70 EO 90

Time (min)

B C

"d

-116

-95

-66 S S .+ -43

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 2

FIG. 2. Reassociation of B peptides with intact spectrin a monomers. A, typical chromatograms using the analytical HPLC gel filtration assay described in Fig. 1 to detect peptide association with the complementary monomers. -, 88 pg of a tryptic digest of 8 monomers in isotonic KC1 buffer ( E / S (w/w) = 1:20,90 min, 0 "C) as a control; - - -, intact a monomers + the tryptic 8 peptides in a 1:2 molar ratio incubated at 0 "C for 24 h prior to injection onto the HPLC. B, SDS-polyacrylamide gel electrophoresis on a 7% gel: lanes 1, 2, and 3, p peptides produced by trypsin digestion at E/S = 1:20, 1:100,1:800 for 90 min at 0 "C, respectively; and lanes 4-12, complexes from a series of experiments between a monomers and bound 8 peptides corresponding to the region indicated by the brackets in A above for: lanes 4, 5, and 6, complexes between 8 peptides (E /S = 1:20) and CY monomers incubated at 0 "C, 0 min; 37 "C, 45 min; 0 "C, 24 h; respectively; lanes 7,8, and 9, complexes between a subunit and 8 peptides ( E / S = 1:lOO) a t 0 "C, 0 min; 37 "C, 45 min; 0 "C, 24 h; respectively; lanes IO, 11, and 12, complexes between a subunit and 8 peptides ( E / S = 1:800) a t 0 "C, 0 min; 37 "C, 45 min; 0 "C, 24 h; respectively. The arrows labeled a-f indicate major 8 peptides which were identified by N-terminal sequencing. Positions of protein stand- ards are indicated on the right in kilodaltons. C , immunoblot (10% gel) using a PIV domain monoclonal antibody. Lane I , 8 peptides from an E/S = 1: lOO digestion (equivalent to lane 2 in B ) ; and lane 2, 8 peptides associated with a monomers (equivalent to lanes 7-9). Bands labeled b, c, and d correspond to those similarly labeled in B. The arrow indicates a 28-kD.a peptide from repeats 5-7 in the BIV domain containing the monoclonal antibody epitope.

described here was that between 10 and 20% of the monomers purified by the 3 M urea, ion exchange method did not refold properly. These improperly folded peptides eluted from the gel filtration column as a very broad peak between the dimer and tetramer regions, could not reassemble into dimers, were more protease-susceptible, and showed increased light scat- tering. The improperly folded monomers could be removed by gel filtration using either conventional chromatography such as a Sephacryl column or by preparative HPLC gel filtration as described under "Experimental Procedures." The experi- ments shown here used monomers purified by preparative HPLC gel filtration. HPLC-purified monomers were stable for at least several weeks without evidence of aggregation when stored at 0 "C in concentrations up to 5 mg/ml, and they retained normal HPLC elution times and the ability to reassemble.

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14778 Nucleation Site for Spectrin Dimer Assembly TABLE I1

Side-to-side reassembly of p peptides with a monomers Peptide locations were determined by either N-terminal sequence analysis or by comparison to previously determined positions on SDS

gels (Speicher et al., 1982; Speicher and Marchesi, 1984). Approximate C-terminal location was based on peptide sizes as estimated from gels. Results are summarized from several different experiments using a range of digestion conditions and different peptide-monomer ratios as described in the text.

(3 peptides" Identification Locationb Domain'

Peptides capable of reassembly T202 ( a ) T192 ( b ) T118 ( c ) T74 ( d ) T46 ( f 1

T65 T33 T28 T22 T17

Peptides which do not reassemble

VIDHAIETEK . . . VIDHAIETEK . . . VIDHAIETEK . . . VIDHAIETEK . . . VIDHAIETEK . . .

2D gels 2D gels 2D gels

ALADEREVVQ . . . 2D eels

293 293 293 293 293

1385 1036 718 47

1934

IV, 111, 11, I IV, 111, I1 IV, 111 IV (repeats 1-7) IV (repeats 1-4)

I1 111 IV ABd I

"These tryptic peptides were named based on apparent molecular mass estimated from SDS gels. Letters in parentheses correspond to

Residue number in the complete p sequence (Winkelmann et al., 1990). Sequences of normal domain peptides identified on two- bands similarly labeled in Fig. 2.

dimensional (2D) gels had been previously determined (Speicher and Marchesi, 1984). e Domain nomenclature as previously described (Speicher et al., 1982).

AB = actin binding site. This region was not identified in the original spectrin domain map.

Reassembly of dimers occurs with high fidelity within sec- onds as shown in Fig. 1, even at low temperatures. In contrast to earlier analysis methods, primarily native gels and ultra- centrifugation studies which have long analysis times (up to 48 h), the gel filtration assay time scale is on the order of minutes rather than days. Separation of different species starts as soon as the sample is applied to the column and even if additional assembly occurs during chromatography, the retention time would be shifted noticeably and the peak might broaden. As shown, when proportional molar amounts of monomers are mixed at 0 "C and immediately injected (within about 10 s), a single major peak in the dimer region (0.10 min later than dimer) is obtained (Fig. 1, panel D ) .

Preservation of a . p Side-to-Side Peptide Complexes during HPLC Gel Filtration-Most spectrin peptides produced by mild trypsin cleavage of spectrin remain associated in large noncovalent complexes during HPLC chromatography. This indicates that extensive side-to-side associations of a and p peptides occur, and these complexes have sufficiently high affinity to remain associated during HPLC gel filtration. As summarized in Table I, the two major peptides that disso- ciated from large noncovalent complexes after mild trypsin treatment ( E / S = 1:200, 0 "C, 90 min) as well as several smaller peptides which remained in the complex were identi- fied by N-terminal sequence analysis. The only peptide that completely dissociated from the complex was the /3 subunit actin binding domain (band b). The major a1 domain peptide, 80 kDa (band a ) , partially dissociated from the complex while all other a and peptides including a 35-kDa a11 domain (band d ) and a 20-kDa (31 domain peptide (band e ) were almost entirely retained in the complex peak. Under the conditions used here, there is only a slight broadening and moderate shift toward later retention times of the noncovalent "tetramer" and "dimer" complexes. As the extent of trypsin cleavage is increased, these peaks continue to broaden and shift toward later retention times, indicating that extensive proteolytic cleavage increases the flexibility of these non- covalent complexes.

Identification of p Peptides Capable of Side-to-Side Reassem- bly-Based on the facile reassembly of heterodimers and retention of extensive noncovalent side-to-side a . p complexes after proteolysis, it was initially expected that most spectrin

peptides would rapidly reassemble side-to-side with the com- plementary subunit. However, initial peptide reassociation experiments showed that many peptides could not reassemble with the complementary subunit (data not shown) even though the same peptides did remain in complexes when spectrin was cleaved with trypsin as described above.

To determine which peptides could reassemble side-to-side, monomer peptide mixtures were combined with intact com- plementary monomers and separated by HPLC chromatog- raphy. One subunit was cleaved with varying E/S ratios of trypsin at 0 "C to produce ladders of peptides ranging from intact subunit to small peptides (see Fig. 2B, lanes 1-3). Various peptide mixtures were combined with the comple- mentary subunit, typically using either equimolar ratios or an excess of peptides. A range of preincubation conditions was tested since recombination of peptides might be slower than reassociation of intact monomers.

Representative results from a series of reassembly experi- ments using p peptides and intact a monomers in a 2:l molar ratio are shown in Fig. 2. Three different f l subunit cleavages (B, lanes 1-3) and the complex peak fractions from recombi- nation of these digests with a monomers using three different preincubation conditions (lanes 4-12) show that only a spe- cific subset of peptides can reassemble. There are slight quan- titative differences in the amounts of bound peptides with different incubation conditions prior to HPLC separation, especially the samples incubated at 37 "C. However, there are no qualitative differences between incubation conditions, the same peptides reassembled under all conditions tested. Reas- sembled peptides were those that showed a specific shift from later eluting positions into the monomer region. These pep- tides were identified by N-terminal sequence analysis or two- dimensional gels as summarized in Table 11.

All p peptides which associated with a monomers contained the N-terminal region of the pIV domain which encompasses the first four homologous segments located immediately after the N-terminal actin binding domain. These four repeats alone were sufficient for high affinity side-to-side association since this 46-kDa peptide assembled with the a subunit even in the presence of larger peptides and when the peptides were present in a molar excess over a subunits (see band f in Fig. 2 B ) . One peptide in the complex peak, band e (sequence,

Page 5: Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side ...

Nucleation Site for Spectrin Dimer Assembly 14779

0 % ::

8 monomer + a peptides """""~"""""""- a peptides alone

0 10 20 30 40 50 60 70 80 90

Time (min)

C

- 80 - D -ar""" -" I -a -43

1 2 3 4 5 6 7 8 9 1 0 1 2 3

FIG. 3. Reassociation of CY spectrin peptides with intact 8 monomers. A, chromatograms of an a subunit peptide mixture from trypsin digestion in isotonic KC1 buffer (E/S (w/w) = 1:100,90 min, 0 "C) as a control, 200 pg (-) and intact @ monomers + the tryptic a peptides in a 1:l molar ratio which was injected onto the HPLC immediately after mixing both components a t 0 "C (- - -). B, SDS- polyacrylamide gel electrophoresis on a 10% gel of the HPLC sepa- ration shown in A. Lane l , the a subunit peptides alone before HPLC separation; lane 2, the a peptide + @ monomer mixture prior to injection onto the HPLC; lanes 3 and 4, fractions from the complex peak marked with an arrow in section A; lanes 5-10, fractions from the remainder of the chromatogram (unbound peptides). The bands labeled a and b, with approximate molecular masses of 52 and 41 kDa, respectively, were the smallest specifically associated a peptides which have been identified. Molecular masses of standard proteins are indicated on the right in kilodaltons. C, immunoblot using an aV domain specific polyclonal antibody. Lane 1, a peptides alone (same as lane 1 in B); lane 2, a peptide-@ monomer complexes (equivalent to lanes 3-4 in B , lane 3, unbound a peptide pool (equivalent to lanes 5-10 in B ) . Bands a and b correspond to those similarly labeled in B.

NLHNKWLKHQ.. ., PI1 domain), did not appear to specifi- cally associate with a spectrin since it is noncovalently com- plexed with band c which does reassemble. The band c + e complex was identified in chromatograms of the peptide mix- ture alone where the two bands co-migrated at an anomalously high molecular weight (data not shown). This suggested that this 68-kDa PI1 domain peptide (band e ) was bound to band c rather than reassembled side-to-side with the a subunit. Also, when more extensive digestions were tested for reassem- bly, a closely related, slightly smaller 65-kDa PI1 peptide did not reassemble with a monomers. Western blotting of com- plex peak fractions using a monoclonal antibody specific for the PIV domain showed that all peptides in the complex, which were 74 kDa (band d ) or larger, reacted with the PIV antibody. Band f , the associated 46-kDa peptide (repeats 1- 4), does not react with this monoclonal which reacts with a 28-kDa peptide (repeats 5-7) as indicated by the arrow in panel C. As shown, the 28-kDa peptide does not reassemble.

Identification of a Peptides Capable of Side-to-Side Reus- sembly-Reassembly of a peptides with intact B monomers was characterized in the same manner as that described above for reassembly of /3 peptides. Similar to the results described

above, reassembly of a peptides with monomers was quali- tatively independent of the different preincubation conditions tested, no preincubation, 0 "C 24 h and 37 "C 45 min (data not shown).

Typical results from the reassembly of a peptides and P monomers at equimolar ratios, which were injected onto the HPLC immediately after mixing, are shown in Fig. 3. The only peptides that specifically associated with /3 monomers in this sample were a 52-kDa and a 41-kDa peptide from the aV domain. The sequences of both bound and unbound peptides from this experiment as well as similar experiments using less extensive cleavages were determined, and the results are summarized in Table 111. These results indicate that all pep- tides capable of reassembly with the /3 subunit contained the aV domain 41-kDa peptide, and all peptides that could not reassemble lacked this region of the molecule. Western blot- ting of both bound and unbound fractions from experiments using a range of digestion conditions confirmed that all pep- tides that could reassemble contained the a V domain. The 52-kDa and 41-kDa a V peptides were the smallest fragments identified that could reassemble side-to-side with /3 monomers although incomplete association of these fragments in the illustrated equimolar recombination experiment suggests a substantially reduced binding affinity (also see below). Since the 41-kDa and 52-kDa peptide share the same N-terminal sequence, the 52-kDa peptide apparently extends into the EF hand region while the 41-kDa peptide does not contain this region. Smaller peptides from the a V domain cannot reassem- ble as indicated by the 25-kDa band from aV that does not bind to /3 monomers (Fig. 3C).

Competition of Peptides and Intact Monomers for Binding to Complementary Subunits-The relative binding affinities of smaller a peptides and intact a monomers for side-to-side association with /3 monomers were evaluated in a series of competition experiments as summarized in Fig. 4. The addi- tion of an equimolar amount of intact a subunits totally abolished association of the a peptides with /3 monomers, while the intact complementary subunits readily reconstituted into heterodimers. In addition, reassembled a V domain pep- tides could be displaced by subsequent addition of intact a monomers (compare Fig. 4C with the control in Fig. 4A).

Reduced binding affinity of the a V domain peptides also occurs in the presence of larger a peptides as illustrated by preferential binding of larger fragments. The Western blot in Fig. 5, using an a V domain specific antibody, summarizes the results of combining a 2-fold molar excess of an a peptide mixture from a very mild trypsin cleavage with /3 monomers. This experiment clearly shows that all fragments larger than about 80 kDa that contain the aV domain region can effec- tively reassemble, while all a V reactive peptides smaller than 80 kDa including the 52- and 41-kDa a V domain peptides are only modestly incorporated into side-to-side a. P complexes. Therefore, both larger peptides and intact a subunits bind to P monomers more strongly than the aV domain alone.

The binding affinity of /3 peptides to intact a monomers also increases with the size of the peptide. However, the smallest P peptide capable of reassembly (PIV T46) has a stronger binding affinity for a monomers than the smallest a peptide (aV T41) binding affinity for the P monomer. This substantially stronger affinity under similar conditions is illustrated by the fact that the PIV T46 peptide is effectively incorporated into side-to-side complexes using a 2-fold molar excess of B peptides (see band f in Fig. 2B), while in a similar, complementary experiment, the CYV T41 is not incorporated into side-to-side complexes (see Fig. 5). The substantially weaker binding of the CYV T41 peptide suggests that the

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14780 Nucleation Site for Spectrin Dimer Assembly

TABLE I11 Side-to-side reassembly of a peptides with p monomers

Peptide locations were determined by N-terminal sequence, and approximate C-terminal locations were based on peptide sizes as estimated from SDS gels. Results are summarized from several different experiments using a range of digestion conditions and different peptide- monomer ratios as described in the text.

m peptides" Identification Location' Domain'

Peptides capable of reassembly T41K ( a ) LQLEDDYAFQ . . . 1921 I T52 K ( b ) LQLEDDYAFQ . . . 1921 V, E P ? T130K REEPGNITQR . . . 1047 111, IV, v T140K REEPGNITQR.. . 1047 111, IV, V, EF? T150K REEPGNITQR . . . 1047 HI, IV, V, EF T200K GTQLHEANQQ . . . 680 11, 111, IV, V, EF

T46K GTQLHEANQQ . . . 680 I1 T52K REEPGNITQR . . . 1047 111 T74K GQKLEDSYHL.. . 46 I (minus 1-39) T8OK ETVVESSGPK . . . 7 I T92K REEPGNITQR.. . 1047 111, IV

Peptides which do not reassemble

"These tryptic peptides were named based on apparent molecular mass estimated from SDS gels. Letters in parentheses correspond to

' Residue number in the complete a sequence (Sahr et al., 1990). bands similarly labeled in Fig. 3.

Domain nomenclature as previously described (Speicher et al., 1982). EF = EF hand motif region. This small protease-sensitive region was not defined in the original spectrin domain map.

a

b

" . -

~ ~- rii " 111)- -68

-a

"b -36

-25

1 2 3 1 2 3 1 2 3

I A A

FIG. 4. Competitive association of a peptides and intact a monomers with intact fl monomers. HPLC fractions are shown on a 10% SDS-polyacrylamide gel from the following experiments: A, 0 monomers (150 pg) were combined with CY peptides alone (150 pg); B, a peptides (150 pg) were mixed with intact a monomers (150 pg) prior to addition of intact 0 monomers (150 pg); C, CY peptides (150 pg) were combined with p monomers (150 pg), incubated 5 min a t 0 "C, then combined with intact a monomers (150 pg). Samples in each panel (120 pl/lane): lane 1, complex peak; lanes 2-3, unbound peptide fractions. Bands labeled a and b are the 52-kDa and 41-kDa CY peptides that specifically associated with 0 monomers, see Fig. 3 for further details. Lower panels: 25 pl/lane of the complex peak fractions on a 7% SDS gel (same as lane 1 on the 10% gel) to show the high molecular weight components.

neighboring a18 segment may also be required for high affin- ity binding to the p subunit.

Effects of Partial Denaturation on Side-to-Side CY. fi Assern- bly-Peptide mixtures were also reconstituted with the com- plementary subunit in isotonic KC1 buffer containing 3 M urea to determine whether additional peptides could reassem- ble side-to-side after partial denaturation of the peptides and subunits. The urea was removed by dialysis, and the samples were analyzed by HPLC gel filtration and gel analysis as described above. The same peptides associated with the com- plementary subunit as in the non-urea experiments, and no additional peptides associated side-to-side in the presence of urea.

1 2 3 4

FIG. 5. Immunoblot (10% gel) analysis of a peptides using an aV domain-specific polyclonal antibody. The 90-min tryptic digests of a monomers in isotonic KC1 buffer were combined with intact monomers in a 2:1 molar ratio a t 0 "C and separated by HPLC gel filtration. Lanes I and 2, a monomer digests alone using E/S = 1:20, 1:100, respectively; lanes 3 and 4, the complex peak containing associated a peptides E/S = 1:20, 1:100, respectively. Bands labeled a and b are the 41-kDa and 52-kDa a peptides that specifically associated with 0 monomers, see Fig. 3 for further details. Molecular masses of standard proteins are indicated on the right in kilodaltons.

DISCUSSION

In this report we analyzed side-to-side assembly of spectrin monomers to form antiparallel heterodimers using a rapid HPLC gel filtration assay. This high affinity association occurs within seconds, even at low temperatures (0-4 "C), and goes to completion even a t very low concentrations. Only some peptide fragments from each subunit could reassemble with the complementary subunit, despite the fact that exten- sive side-to-side CY.@ complexes were retained if dimers were cleaved with proteases such as trypsin.

The locations of peptides that could associate (Fig. 6) indicate that heterodimer assembly is initiated a t a single, discrete region on each subunit located near the actin binding end of the flexible, rod-like dimer. Both regions, which will be referred to as heterodimer nucleation sites, are indicated by hatched boxes in Fig. 6. The adjacent EF hand region on the CY subunit and the adjacent actin binding domain on the fi subunit are not involved in assembly. One of the most striking features concerning the nucleation regions is that three of the repeats (two in the CY and one in the p subunit)

Page 7: Properties of Human Red Cell Spectrin Heterodimer (Side-to-Side ...

Nucleation Site for Spectrin Dimer Assembly 14781

t I 200K

t I 150%

I I 1 130K

I 140K

1-1 52K

I-{ 4 l K

NUCLEATION SITE

DOMAINS t I ,, I I ,I

eo " 46 'I

111 I,

52 " I V 4 1 I

N I I I I 20 ALPHA l,l,l h

C I 1151 I I I 1101 UmN BETA

e WMAINS

NUCLEATION SITE

1-1 74K

1-1 46K

1-1 l l 8 K

1 192K I t 1 202K

FIG. 6. Model of spectrin structure illustrating the side-to- side nucleation site. The various structural motifs of the antipar- allel 01 and 0 subunits are schematically represented rectangles, repetitive segments (repeats with an 8-residue insert have an added triangle on their sides); square, src SH-3 type segment; diamonds, EF hands; large rectangle, actin binding domain; triangle, nonhomologous phosphorylated region. Repetitive segments are numbered starting from the N-terminal ends of the subunits. Peptide domains are numbered from the tetramerization site with Roman numerals as previously described (Speicher et al., 1982), and the EF hand domain (EF) and actin binding domain (AB) deduced from the complete cDNA sequences have been added. Numbers below domain bars are approximate molecular masses of the largest unique domain peptide. Locations of a peptides that reassemble with 0 monomers are shown above the model, and p peptides that reassemble are shown below the model.

have an 8-residue insertion in the normal 106-residue repeat unit. In most spectrin repeats, the segment length of 106 residues is very precisely preserved and only a few repeats have insertions or deletions. Therefore, these 8-residue inser- tions might confer unique, conformational properties upon the nucleation sites which are responsible for the specific initial association. The nucleation regions also contain re- peats most homologous to those in a-actinin as previously noted for Drosophila spectrin (Dubreuil et al., 1989). The a- actinin molecule contains only four spectrin-like repeats, and these repeats are responsible for forming strong noncovalent associations in the antiparallel a actinin homodimer (Ima- mura et al., 1988).

In an earlier study, a series of PIV domain peptides were also tested for their ability to reassemble with intact 01 mon- omers (Sears et al., 1986). These peptides were isolated on a calmodulin affinity column in the presence of 6 M urea. Consistent with the present study, it was observed that the PIV domain 74-kDa peptide could form high affinity com- plexes with the a subunit. However, this previous study con- cluded that smaller PIV domain peptides, including the 46- kDa peptide identified in this study as the minimum nuclea- tion site, could not reassemble. There are two likely expla- nations for this discrepancy. First, the smaller peptides may have been irreversibly denatured in the 6 M urea used during calmodulin affinity column purification in the earlier study. Alternatively, assembly of these smaller peptides may have been overlooked in the earlier study since the migration position of the PIV 52- and 46-kDa peptides on control native gels was quite close to the position of the complexes and association of these peptides with a monomers could not be clearly discerned (see Fig. 6 in Sears et al., 1986).

The complementary locations of the a and P nucleation sites in the schematic model, shown in Fig. 6, as well as the homology of this region to a-actinin, strongly suggest that the a and nucleation sites bind directly with each other. Fur-

thermore, the unique conformation of these repeats probably produces a specialized lock and key type of conformation in the monomer state that initiates assembly and defines the register of the complementary repeat units in the two spectrin subunits. The nucleation site is therefore not only responsible for initial a.P binding, but also controls the side-to-side register or phasing of the many homologous repeats in both subunits. As illustrated in Fig. 6, the a21 segment would interact directly with the D l segment, a20 associates with P2, etc. The lock and key nucleation model would explain why staggered side-to-side associations of the two subunits do not occur as shown by electron microscopy of reconstituted dimers (Yoshino and Marchesi, 1984).

Although the strongest side-to-side associations of mono- mers may occur at the physical ends of the dimer rod-like structure as suggested by electron microscopy (Shotton et al., 1979), it is clear that many additional side-to-side a . p asso- ciations occur along the length of the dimer (Table I and Morrow et al., 1980). In the assembled heterodimer, each homologous repetitive segment probably associates side-to- side with a specific counterpart segment of the complementary subunit along the length of the dimer. A reasonable, hypo- thetical mechanism for side-to-side association of spectrin monomers would resemble a zipper. After initial association of the nucleation sites, a conformational change is propagated along the full length of both chains, which permits each subsequent segment to pair with its unique partner on the complementary subunit. This propagation step probably in- volves supercoiling of the two subunits to form a twisted, rope-like structure as has been proposed by Fourier analysis of electron micrographs (McGough and Josephs, 1990). I t is unlikely that the reconstituted peptide-monomer complexes only associate at the nucleation site, since there is a progres- sive retention time shift of reassembled complexes from the monomer position toward the dimer position proportional to the size of the reassembled peptides. Since reassembled dimers are less flexible than monomer, this proportional length- related decrease in flexibility could only occur if the associated peptide assembles along its entire length. Also, peptide-mon- omer complexes tethered at a single site would hydrodynam- ically resemble head-to-head monomer-peptide complexes. Since head-to-head peptide-monomer complexes can be clearly resolved from side-to-side complexes (data not shown), it is very likely that the side-to-side complexes are assembling along their entire lengths.

Identification of these nucleation sites as the controlling element in heterodimer assembly and their location is highly consistent with a recent report of an aIV-V junction poly- morphism which affects polymorphic allele incorporation into the membrane skeleton (Alloisio et al., 1991). Considering the data presented herein, it is likely that the aIV-V mutation directly affects the binding affinity of the nucleation site and hence influences the relative efficiency of incorporation of the affected allele into the mature membrane skeleton.

Acknowledgments-We thank Kevin S. Beam and Amy Scally for technical assistance, Lauren Kim for editorial assistance, and Kaye D. Speicher for computer analyses.

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