From the Departments of Pathology and
¶ Pediatrics, Yale University School of Medicine,
New Haven, Connecticut 06510
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ABSTRACT |
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The ankyrin 33-residue repeating motif, an
L-shaped structure with protruding -hairpin tips, mediates specific
macromolecular interactions with cytoskeletal, membrane, and regulatory
proteins. The association between ankyrin and
-Na,K-ATPase, a
ubiquitous membrane protein critical to vectorial transport of ions and
nutrients, is required to assemble and stabilize Na,K-ATPase at the
plasma membrane.
-Na,K-ATPase binds both red cell ankyrin
(AnkR, a product of the ANK1 gene) and
Madin-Darby canine kidney cell ankyrin (AnkG, a product of
the ANK3 gene) utilizing residues 142-166
(SYYQEAKSSKIMESFK NMVPQQALV) in its second cytoplasmic domain. Fusion
peptides of glutathione S-transferase incorporating these
25 amino acids bind specifically to purified ankyrin
(Kd = 118 ± 50 nM). The
three-dimensional structure (2.6 Å) of this minimal ankyrin-binding motif, crystallized as the fusion protein, reveals a 7-residue loop
with one charged hydrophilic face capping a double
-strand. Comparison with ankyrin-binding sequences in p53, CD44, neurofascin/L1, and the inositol 1,4,5-trisphosphate receptor suggests that the valency
and specificity of ankyrin binding is achieved by the interaction of
5-7-residue surface loops with the
-hairpin tips of multiple
ankyrin repeat units.
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INTRODUCTION |
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Tethering interactions between the cytoplasmic domains of integral membrane and other proteins, mediated by ankyrin or proteins containing ankyrin-like repeat structures, play fundamental roles in diverse biological activities including growth and development (1-5), intracellular protein trafficking (6-8), the establishment and maintenance of cellular polarity (9-12), cell adhesion (13, 14), signal transduction (2, 15-19), and mRNA transcription (20, 21). Ankyrin, including its many isoforms (reviewed in Refs. 22 and 23), is also the most ubiquitous adapter protein mediating linkage of membrane proteins with the spectrin-based skeleton, both at the plasma membrane (reviewed in Refs. 23-25) as well as with internal membrane compartments including the Golgi apparatus (6-8). A characteristic feature of ankyrin is the presence of well conserved 33-residue repetitive units (ankyrin repeats) that individually or in combination bind to transmembrane proteins (reviewed in Ref. 23). Because no data on the structure of an ankyrin-binding domain in an integral membrane protein exist, it is unclear how so many diverse membrane proteins can bind specifically to a single ankyrin molecule (26, 27).
The -subunit of Na,K-ATPase interacts with ankyrin (11, 28). This
interaction stabilizes Na,K-ATPase at the plasma membrane and enables
its transport from the endoplasmic reticulum to the Golgi (8).
Cytoplasmic domains (CD)1 II and
III of
-Na,K-ATPase bind ankyrin in vitro. These
sequences appear to act independently, and those in CDII (residues
140-290) account for most of the affinity of
-Na,K-ATPase for
ankyrin (28, 29). We now identify a 25-residue minimal ankyrin-binding (MAB) sequence within CDII of
-Na,K-ATPase and report its
three-dimensional structure. This peptide forms a loop-on-a-stalk motif
well suited for interaction with one or more of the putative
-hairpin tips of the ankyrin repetitive unit (5). We anticipate that
the ankyrin-binding structure identified here will emerge as a common feature of other ankyrin-binding proteins. Portions of this work have
been presented in abstract (30).
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MATERIALS AND METHODS |
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Preparation and Purification of Recombinant
Peptides--
Oligonucleotides were designed to amplify constructs
IIa, IIb, and IIc from a construct encoding CDII of -Na,K-ATPase
(28). Amplification products were subcloned into TA vectors
(Invitrogen) and sequenced by the dideoxynucleotide chain termination
method (U. S. Biochemical Corp.). Constructs were expressed as
fusions with Schistosoma japonicum glutathione
S-transferase (SjGST) using the pGEX-2T prokaryotic
expression vector (Amersham Pharmacia Biotech) and purified using
glutathione-Sepharose (28). SjGST was expressed as a control peptide.
All peptides were eluted with 50 mM Tris-HCl, 5 mM reduced glutathione, pH 8.0, and dialyzed into
ankyrin-binding buffer (ABB; 50 mM Tris-HCl, pH 6.9, 50 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 1 mM EGTA, 1 µM phenylmethylsulfonyl fluoride, 1 mM Pefabloc SC). Proteins were analyzed by
SDS-PAGE.
Ankyrin Binding Assay-- Each fusion protein (50 µg at 1 µg/ml) was conjugated to 50 µl of a 50% slurry of glutathione-agarose for 1 h at 4 °C with gentle rotation. Ankyrin (AnkR) was purified by extraction of spectrin-depleted fresh erythrocyte inside-out vesicles with 1 M KCl, followed by ion exchange chromatography on DEAE cellulose (31). AnkR binding was assayed by adding 25 µg to the peptide-conjugated beads in a total volume of 500 µl in ABB, overnight incubation at 4 °C, and analysis of the bead fraction by SDS-PAGE. Ankyrin was detected by Western blotting with specific antibodies (28). Confluent MDCK cells extracted in situ were used to prepare a high salt extractable cytoskeletal fraction (Fx2) enriched in MDCK cell ankyrin (AnkG) (28). Conjugated beads were incubated with Fx2 (300 µg of total protein), and bound ankyrin was detected as above. Other procedures and antibodies were as before (7, 28).
For quantitative binding measurements, purified ankyrin was labeled with sulfo-N-hydroxysuccinimide biotin (Pierce), further purified by gel filtration, and concentrated using a Centricon ultradialysis membrane (7). Aliquots (10 µg) of purified fusion peptide IIa (GST-MAB) or GST were incubated as above in ABB with varying concentrations of biotinylated ankyrin. Bound ankyrin was visualized by ECL after direct overlay with horseradish peroxidase-avidin (Vector). Relative binding was measured by densitometry of autofluorograms, with precautions taken to assure linearity of detection. Because the absolute free concentration of ankyrin could not reliably be measured using this biotinylated assay, free ankyrin was assumed to equal total ankyrin. This approximation will systematically overestimate slightly the Kd (weaker apparent affinity than the true value). Nonspecific binding to GST was subtracted prior to nonlinear regression fitting. Errors are expressed as ± 2 S.D..Structure Determination--
Fusion peptide IIa was purified by
high pressure liquid chromatography gel filtration into 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 2 mM -mercaptoethanol. Crystals were prepared at room
temperature by hanging drop vapor diffusion against a reservoir of 30%
polyethylene glycol 4000, 100 mM bis-tris-propane, 150 mM NaCl, 40 µM
-mercaptoethanol, pH 8.8 (32). Diffraction data were collected with a R-AXISII imaging plate
detector mounted on a Rigaku 200HM generator at
170 °C using a
crystal flash frozen in crystallization buffer. Data were processed
using the program DENZO and SCALEPACK (33) and were 86% complete
(>2
). The structure was determined by molecular replacement using
the published crystal structure of GST from S. japonica (34)
and the program AMORE (35) of the CCP4 program suite (33). Successive
rounds of model building and simulated annealing refinement were
carried out with the program X-PLOR version 3.851 (36) and the CCP4
program suite. The graphic display program O, version 5.10 (37) was
used to build and correct the structure manually. Each residue of the
final model was checked by the omit map.
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RESULTS AND DISCUSSION |
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The Minimal Ankyrin-binding Domain of
-Na,K-ATPase--
Recombinant peptides derived from CDII of rat
-Na,K-ATPase (28) were prepared as fusion proteins with GST and
assayed for their ability to bind AnkR (from red cells) or
AnkG (from Madin-Darby canine kidney cells) (Fig.
1). All peptides were of the predicted molecular mass, soluble, and readily purified (Fig. 1B).
Peptide IIa (residues 142-166 of
-Na,K-ATPase numbered according to
Ref. 38) retained full binding activity to both AnkR and
AnkG and constituted the MAB sequence. CDII peptides not
encompassing MAB, such as peptide IIc (residues 168-286), were devoid
of activity.
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GST-MAB Binds Ankyrin with High Affinity--
Prior studies have
detected Na,K-ATPase affinities for ankyrin in the range of 50-2600
nM (11, 39, 40). Of interest was whether GST-MAB bound
ankyrin with comparable affinity. Purified ankyrin (AnkR)
was biotinylated, and its binding to GST-MAB or GST was measured (Fig.
2). Nonlinear regression analysis based on a
bi-molecular binding model indicated that ankyrin specifically bound
GST-MAB with a Kd = 118 ± 50 nM,
in agreement with the affinity of intact Na,K-ATPase for ankyrin. Thus,
although other regions of -Na,K-ATPase such as the ALLK motif
identified in CD3 may contribute to its interaction with ankyrin (28,
29), the 25 residues identified here are sufficient and bind
specifically to ankyrin but not to other proteins present in the whole
kidney lysates (Fig. 1). GST alone was devoid of significant binding activity (Fig. 2). In separate studies we have also demonstrated that
loss of these 25 residues in
-Na,K-ATPase abrogates its binding to
ankyrin in vivo (41).
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Structure Determination of MAB--
Attempts to crystallize
-Na,K-ATPase CDII or MAB alone were not successful. Because the
GST-MAB fusion peptide contained an ankyrin-binding domain that was
fully active, carrier-mediated crystallization was used to obtain
structural information (32). The active GST fusion peptide containing
MAB (construct IIA, Fig. 1, and Fig. 3)
yielded ordered crystals (space group of P4(3)2(1)2, with
a = b = 92.17 and c = 57.57). Diffraction of this crystal at
170 °C yielded a data set
that was 86% complete (>2
) and allowed the determination of the
complete structure of the GST-MAB fusion peptide with an
R-factor of 19.8 and an Rfree of 36.5 (1% of total data) at 2.6 Å resolution (Fig. 3), using a molecular replacement strategy based on the known crystal structure of S. japonica GST (SjGST) (which has one molecule in the asymmetric unit and 41% solvent) (34). The refined model displayed a continuous main chain electron density and consisted of two domains, one representing SjGST and the other MAB (Fig. 3). Each residue of MAB was
verified with an electron density omit map. Both the GST and MAB
domains were well defined when the total structure was checked with the
2Fo
Fc map, including
the side chains of Leu118, His215, and
Lys218, residues disordered in the published structure of
SjGST (34). Backbone residues 1-210 of SjGST superimposed on those of
SjGST-MAB with a RMS deviation of 1.02 Å. Differences between SjGST
and SjGST-MAB were found in the position of Met1 and the
positions of residues 211-218, which flank a region of disorder in
SjGST (but not in SjGST-MAB). There were no discernible hydrophobic or
salt interactions between GST and MAB. A single H-bond was detected
between Arg224 (residue 4 of the linker sequence) and
Gln248 (MAB, corresponding to Gln163 in
-Na,K-ATPase); this bond does not alter the SjGST backbone conformation relative to SjGST alone. The three Gly residues (at positions 211, 212, and 213) in GST, together with two Pro residues at
positions 216 and 217 and the
Pro223-Arg224-Gly225-Ser226
of the linker sequence (linker positions 3-6) collectively appear to
well insulate MAB from secondary structural perturbations arising in
SjGST. The crystal structure of the fusion peptide thus displays two
well separated and independently folded motifs, lending confidence that
the structure of MAB as revealed in the fusion protein crystal is
valid. Finally, it is clear from the graphical display that four MAB
units are packed in each unit cell. Although theoretically the MAB
conformation might be influenced by the packing force, these forces are
usually quite small. Taken together, these considerations argue
strongly that the conformation of MAB will not be influenced by the
presence of SjGST and that carrier-mediated crystallization of active
ankyrin-binding peptides fused with SjGST may represent an important
and general approach to identifying the structural determinants of
ankyrin binding activity in a variety of proteins.
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The Loop-on-a-Stalk Structure of MAB--
Detailed analysis
of the ankyrin-binding domain within SjGST-MAB reveals an antiparallel
double -strand flanking a loop composed of the seven residues
MESFKNM (residues 152-158 of
-Na,K-ATPase) (Fig.
4). The overall structure of MAB is
suggestive of a loop on a stalk. This loop is amphipathic, presenting a
hydrophobic face composed of two methionines and one phenylalanine and
a hydrophilic face composed of Glu-Ser and Lys-Asn on the opposite
side. A dipolar interaction between Ser149 (position 234 in
the crystal) and Gln163 (crystal position 249) of the
antiparallel
-strands stabilizes the stalk. Presumably, in intact
-Na,K-ATPase, flanking sequences would further stabilize the stalk
and probably alter the positioning of the terminal residues in MAB. A
search of nucleotide and protein sequence banks (GenBankTM,
Swiss-Prot) revealed exceptional conservation of MAB across species and
between isoforms of
-Na,K-ATPase (Fig. 5).
Sequences partially homologous to MAB also exist in gastric H,K-ATPase
(42), which associates with ankyrin in gastric parietal cells (43). No
homologous sequences were noted in other well documented
ankyrin-binding proteins (including the erythrocyte anion exchanger,
the amiloride-sensitive sodium channel, the voltage-sensitive sodium
channel, the Na+/Ca2+ exchanger, CD44,
neurofascin, and IP3-R).
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Curristin, Dr. C. Cianci, and C. Traub for assistance with the purification of ankyrin.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
Supported by grants from the National Institutes of
Health.
** To whom correspondence should be addressed: Dept. of Pathology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-3624; Fax: 203-785-7037; E-mail: jon.morrow{at}yale.edu.
1 The abbreviations used are: CD, cytoplasmic domain; MDCK, Madin-Darby canine kidney; IP3, inositol 1,4,5-trisphosphate; IP3-R, IP3 receptor; MAB, minimal ankyrin-binding; GST, glutathione S-transferase; SjGST, S. japonica GST; ABB, ankyrin-binding buffer; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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