From the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
Received for publication, October 30, 2002, and in revised form, December 6, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cytoplasmic domain of erythrocyte
membrane band 3 (cdb3) serves as a center of membrane organization,
interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, a tyrosine phosphatase, and a
tyrosine kinase, p72syk. The crystallographic structure of the
cdb3 dimer has revealed that residues 175-185 assume a Ankyrin mediates the attachment of a diverse set of membrane
spanning proteins to spectrin-based membrane skeletons. Depending on
the cell type, ankyrin may bridge between the Ankyrin is folded into three independent domains that include an 89-kDa
NH2-terminal membrane-binding domain, followed by a 62-kDa
spectrin-binding domain and a COOH-terminal regulatory domain. The
membrane-binding domain of ankyrin consists of 24 tandem repeats of a
33-amino acid motif known as the ankyrin repeat that is involved
in protein recognition (11-14). Because ankyrin interacts with a
highly diverse group of membrane proteins, much effort has been devoted
to identifying the structural features that mediate these
interactions (15-18).
The major linkage between the membrane bilayer and spectrin-based
cortical skeleton in erythrocytes is mediated by ankyrin binding to
band 3. Because previous studies (19-22) aimed at mapping the
docking site(s) of ankyrin on the cytoplasmic domain of erythrocyte membrane band 3 (cdb3)1 were
conducted without the benefit of the crystal structure of cdb3, these
investigations of necessity led to inexact conclusions regarding
regions or linear sequences of cdb3 implicated in ankyrin binding.
Thus, modification of Cys-201 and/or Cys-317 of the cytoplasmic domain
of band 3 was found to compromise ankyrin binding (19). Monoclonal
antibodies against residues 190-203 (20) or 174-186 (21) were also
shown to block the ankyrin interaction. Ankyrin association was further
found to protect residues 175-186 of cdb3 from proteolysis (21), and
chimera analysis also implicated participation of residues
155-195 (22).
Similar studies have suggested a contribution from the NH2
terminus of cdb3 in the ankyrin interaction (20). Evidence for NH2-terminal involvement has come from the observation that
kidney cdb3, which lacks residues 1-65, exhibits no affinity for
ankyrin (23, 24). In addition, ankyrin association inhibits
phosphorylation of tyrosine residues (predominantly Tyr-8) at the
NH2 terminus of band 3 (20). It would appear from these
considerations that ankyrin may associate with two disparate regions of
cdb3, a region near residues 175-190 and sequences near the
NH2 terminus.
With the recent publication of the crystal structure of cdb3 (25), it
has become possible to ask more precise questions regarding the docking
site of ankyrin on cdb3. In this study, we have noted that a stem-loop
structure, which is conformationally similar to a proposed
ankyrin-binding site on the Na+/K+-ATPase (26),
is located within the general region defined by the earlier mapping
studies of Willardson et al. (20) and Davis et
al. (21). We have, therefore, decided to evaluate whether this
stem-loop structure might constitute a critical conformational motif
involved in ankyrin-band 3 association. Because of previous evidence
for NH2-terminal involvement, we have also explored the nature of the participation of the region in ankyrin binding in greater detail.
Oligonucleotide-directed
Mutagenesis--
Oligonucleotide-directed mutagenesis was performed
using a QuikChange mutagenesis kit (Stratagene) according to the
manufacturer's instructions. The following oligonucleotides were
synthesized and used for site-directed mutagenesis. For deletion of
amino acids 175-185 and substitution of 2 glycines in their place,
5'-ggccctggggggtgtgaagggtggacagcctctgctcccccaac3-' (2 glycine residues are underlined). A His6 tag was introduced at the COOH terminus of cdb3 in a pT7-7 plasmid (27) and used as the
template. For deletion of amino acids 1-50, PCR was performed using
two primers: forward,
5'-ggaattccatatgcacccgggtacccacgaggtc3-' (start codon and
amino acids 51 are underlined); reverse,
5'-agaaagctttcagtggtggtggtggtggtggaagagctggcctgtctgctg3-' (stop codon and 6 histidine residues are underlined). The resulting mutant cDNAs were sequenced to verify mutations. Plasmids were then
transferred into BL21 (DE3) pLysS cells for expression of the mutated
cdb3 proteins.
Protein Expression and Purification--
Both wild type and
mutant cdb3s with a COOH-terminal His tag were expressed in the pT7-7
bacterial expression system using isopropylthiogalactoside induction
for 3 h at 37 °C. The His-tagged proteins were purified by
Ni-affinity chromatography (Qiagen). Intact ankyrin was purified and
radiolabeled using 125I-Bolton-Hunter reagent as described
by Bennett (28). The 46.5-kDa fragment of ankyrin (residues 403-827)
was expressed in Escherichia coli strain BL21 (DE3)/pLysS
(the expression vector was a kind gift of Dr. Vann Bennett) and
purified as described by Davis and Bennett (29). The GST-tagged 46.5 kDa ankyrin construct was kindly provided by Dr. Peter Michaely at the
University of Texas Southwestern, and the GST-tagged protein 4.1 construct was a generous gift of Dr. Narla Mohandas. GST-tagged
proteins were purified using a glutathione-Sepharose column (Amersham Biosciences).
Structural Characterization of cdb3--
The pH dependence of
the Stokes radius of cdb3, which increases by more than 11 Å between
pH levels 6.5 and 9.5, was evaluated as previously reported (30, 31),
except the fast protein liquid chromatography gel filtration column
used consisted of prepacked Superdex 200HR 10/30 (Amersham
Biosciences). The pH dependence of intrinsic fluorescence emission of
cdb3, which more than doubles between pH levels 6.5 and 9.5, was
measured at 340 nm in solutions of 50 mM sodium borate, 50 mM sodium phosphate, 70 mM NaCl adjusted to
desired pH levels using an Aminco-Bowman luminescence spectrometer at
an excitation wavelength of 290 nm and slit widths set at 6 nm.
In Vitro Protein-binding Assay--
Purified His-tagged wild
type or mutant cdb3 was mixed with increasing concentrations of ankyrin
at 4 °C overnight in 7.5 mM phosphate buffer containing
10% sucrose, 90 mM KCl, 10 mM imidazole, 0.4 mM phenylmethylsulfonyl fluoride, and 1 mg/ml bovine serum albumin at pH 7. Pre-equilibrated Ni-NTA beads were incubated with the
mixture for 30 min at 4 °C and washed six times with the same
buffer. The bound complexes of cdb3-ankyrin were eluted with 200 mM imidazole. The quantity of bound ankyrin was then evaluated by either a quantitative dot-blot assay using an anti-ankyrin antibody, or by measuring the radioactive counts in the eluate whenever
125I-Bolton-Hunter-labeled ankyrin was employed, or by
measuring GST activity for GST-ankyrin fusion constructs. The GST
substrate (100 mM potassium phosphate buffer, pH 6.5/1
mM 1-chloro-2,4-dinitrobenzene/1 mM reduced
glutathione) was added to the eluants and the absorbance at 340 nm was
recorded to determine the amount of bound GST-ankyrin (32). To ensure
that equivalent amounts of His-tagged wild type and mutant cdb3 were
bound to the Ni2+ beads, eluants were assayed for bound
cdb3 using an anti-cdb3 antibody. Binding of GST-30 kDa 4.1 (a
GST fusion construct of the cdb3-binding domain of protein 4.1)
to cdb3 was determined by the same method, except the pH of the
incubation buffer was adjusted to pH 7.4.
Peptide Inhibition of Ankyrin Binding--
Peptides
corresponding to residues 175-185 of human cdb3 were synthesized by
SynPep (Dublin, CA). An additional cysteine was added to the
NH2 and COOH termini of the peptide for subsequent formation of a disulfide bridge between the ends of the peptide in an
effort to mimic the hairpin loop seen in the crystal structure. Increasing concentrations of peptide were then mixed with ankyrin prior
to addition of cdb3, and the binding assay was performed as described above.
Characterization of a Deletion Mutant of cdb3 Lacking a
To confirm that the conformation of the above deletion mutant was not
globally perturbed, the reversible pH-dependent
conformational change characteristic of native cdb3 was examined (30,
31). Thus, as the pH is raised from 6.5 to 9.5, the Stokes radius of native cdb3 enlarges by more than 11 Å and the intrinsic fluorescence, which is highly quenched at lower pH, more than doubles. As seen in
Fig. 2A, titration of the
deletion mutant from pH 6.5 to 9.5 yields the expected fluorescence
increase of 2-fold. Further, comparison of the Stokes radius increase
of the mutant and wild type cdb3 as a function of increasing pH reveals
the same dimensional changes in both proteins (Fig. 2B).
These data demonstrate that the deletion mutant retains the same
structural properties as wild type cdb3.
To further establish that truncation of the Evaluation of the Affinity of Ankyrin for the
Although previous studies have demonstrated that 46.5 kDa ankyrin can
bind cdb3 with high affinity (29) and even drive band 3 into tetramers,
much like intact ankyrin (34), the question still remained whether the
full-length 220 kDa ankyrin, with its twelve additional 33 amino acid
repeats, might be less dependent on the
To further support the hypothesis that residues 175-185 participate
directly and prominently in ankyrin binding, the 11 amino acid peptide
corresponding to residues 175-185 was synthesized, only in this case a
cysteine residue was added to each end of the peptide to allow for
disulfide-mediated cyclization of the peptide. Cyclization was
considered important as a means of trying to mimic the 3-dimensional
structure of the Evaluation of Ankyrin Binding to the NH2 Terminus of
cdb3--
A second ankyrin-binding site proposed in the literature is
thought to reside at the NH2 terminus of cdb3 (25, 37).
Evidence for this contention originally arose from three key
observations: i) kidney cdb3, which lacks residues 1-65, shows no
affinity for ankyrin (23, 24); ii) phosphorylation of cdb3 on tyrosine 8 is inhibited upon addition of ankyrin (20); and iii) antibodies to
the extreme NH2 terminus of cdb3 block ankyrin binding
(20). However, with the advent of the crystal structure of cdb3 (25), the interpretation of at least one of these observations no longer seems so straightforward because deletion of residues 1-65 of cdb3
removes the central strand of a large
In contrast to our expectations from previous studies (20, 23, 24),
cdb3 lacking residues 1-50 exhibited the same binding affinity for
46.5 kDa ankyrin as wild type cdb3 (Fig. 3), suggesting that the
NH2 terminus may not be essential for ankyrin association. Nevertheless, because the previous studies were conducted with intact
ankyrin rather than truncated 46.5 kDa ankyrin, and because the earlier
data still seemed compelling, it was decided to re-evaluate the
affinity of the 1-50 deletion mutant of cdb3, but this time for the
full-length ankyrin (i.e. the 220-kDa ankyrin might still require the NH2 terminus of cdb3). However, as seen in Fig.
4, similar results were again obtained. Finally, to assure ourselves that the previously published studies were not flawed, we repeated the
experiments using 46.5 kDa ankyrin instead of intact ankyrin and found
that all three lines of evidence for NH2-terminal
involvement could be replicated (i.e. kidney band 3 does not
bind 46.5 kDa ankyrin, 46.5 kDa ankyrin inhibits tyrosine
phosphorylation at the NH2 terminus of cdb3, and antibodies
to the NH2 terminus of cdb3 block 46.5 kDa ankyrin binding
(data not shown)). These results argue strongly that although residues
1-50 of cdb3 may not physically participate in ankyrin binding, they
must reside sufficiently proximal to the ankyrin-binding site on cdb3
that antibody binding to the NH2 terminus prevents ankyrin
association, and ankyrin association at the We have presented evidence that the Deletion mutagenesis of the NH2 terminus of cdb3 further
demonstrated that the first 50 residues of the polypeptide are not directly involved in ankyrin binding. However, the observations that
blocking of this sequence by peptide-specific Fabs totally prevents ankyrin binding, and ankyrin binding to the The absence of a requirement for residues 1-50 in ankyrin binding
raises the question of why kidney cdb3, which lacks residues 1-65,
fails to bind ankyrin. Analysis of the crystal structure, however,
demonstrates that residues 57-65 constitute a central strand in an
eight-stranded It is interesting to note that the binding properties of
intact ankyrin to band 3 are reasonably well replicated by the binding properties of 46.5 kDa ankyrin to cdb3. Not only do both polypeptides require association at the Comparison of the proposed Na+/K+-ATPase and
cdb3-binding sites for ankyrin might suggest that a -hairpin
loop similar to a putative ankyrin-binding motif at the cytoplasmic
surface of the Na+/K+-ATPase. To test
whether this hairpin loop constitutes an ankyrin-binding site on cdb3,
we have deleted amino acids 175-185 and substituted the 11-residue
loop with a Gly-Gly dipeptide that bridges the deletion without
introducing strain into the structure. Although the deletion mutant
undergoes the same native conformational changes exhibited by wild type
cdb3 and binds other peripheral proteins normally, the mutant exhibits
no affinity for ankyrin. This suggests that the exposed
-hairpin turn indeed constitutes a major ankyrin-binding site on
cdb3. Other biochemical studies suggest that ankyrin also docks at the
NH2 terminus of band 3. Thus, antibodies to the
NH2 terminus of cdb3 block ankyrin binding to the cdb3, and
ankyrin binding to cdb3 prevents p72syk phosphorylation of cdb3
at its NH2 terminus (predominantly at Tyr-8).
However, a truncation mutant of cdb3 lacking the
NH2-terminal 50 residues displays the same binding affinity
as wild type cdb3. These data thus suggest that the
NH2 terminus of cdb3 is proximal to but not required for
the cdb3-ankyrin interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of spectrin
and the anion exchanger (1), the Na+/K+-ATPase
(2, 3), a voltage-dependent Na+ channel (4), or
the Na+/Ca2+ exchanger (5). Cell adhesion
molecules such as CD44 (6) and L1CAM family members (7, 8), as well as
calcium-release channels such as IP3 receptor (9) and ryanodine
receptor (10) are also known to associate with ankyrin.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hairpin
Loop (Residues 175-185)--
As noted in the Introduction, a proposed
ankyrin-binding site on the cytoplasmic pole of the
Na+/K+-ATPase (26) appears structurally similar
to a
-hairpin loop revealed in the crystal structure of cdb3 (Fig.
1). To evaluate whether this hairpin loop
comprising residues 175-185 of cdb3 might serve as an ankyrin-binding
site, the residues forming the hairpin loop were deleted and a
diglycine bridge was substituted in their place. Thus, based on the 4.9 Å distance separating amino acids 175 and 185 in the crystal
structure, two glycines were calculated to optimally span the gap
without introducing strain into the mutated protein molecule. This
deletion mutant was then expressed in E. coli and purified
to homogeneity for further characterization.
View larger version (61K):
[in a new window]
Fig. 1.
The structure of a cdb3 monomer with the
-hairpin loop comprising residues
175-185 highlighted.
View larger version (12K):
[in a new window]
Fig. 2.
A, comparison of the pH dependence of
the intrinsic fluorescence of purified recombinant wild type and mutant
(residues 175-185 replaced with a diglycine bridge) human cdb3. Both
wild type and mutant cdb3 were dissolved in 50 mM sodium
phosphate, 50 mM sodium borate, 70 mM NaCl,
pre-adjusted to the desired pH. The relative magnitude of the
fluorescence emission at 334 nm ( ex, 290 nm) is plotted
as a function of pH. B, comparison of the change in Stokes
radius of recombinant wild type and mutant cdb3 with pH. Purified wild
type and deletion mutant cdb3 (1 mg/ml) were exchanged into 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, 1 mM EDTA, 0.2 mM
dithiothreitol, and 1 mM NaN3 adjusted to the
desired pH values. The protein samples (200 µl) were then
chromatographed on a prepacked Superdex 200 HR 10/30 column (10 × 300 cm) pre-equilibrated in the same buffer. The Stokes radii were
calibrated as described previously (27). C, comparison of
GST-30 kDa protein 4.1 binding to wild type and mutant cdb3. His-tagged
wild type or mutant cdb3 were incubated with increasing amounts of
GST-30 kDa protein 4.1 at 4 °C overnight in isotonic phosphate
buffer, pH 7.4. The His-tagged cdb3-GST-30 kDa protein 4.1 complexes
were then collected on Ni-NTA agarose beads for 30 min, and the beads
were washed 6 times with 6 volumes of buffer and eluted with 250 mM imidazole. GST activity associated with the pelleted protein 4.1 was then measured by
following absorbance at 340 nm for 1 h at room temperature upon
addition of 1 mM 1-chloro-2,4-dinitrobenzene/1
mM reduced glutathione.
-hairpin loop causes
only a localized change in cdb3 structure, the interaction of cdb3 with
protein 4.1, a second major peripheral protein ligand of cdb3 was
examined. For this purpose, recombinant GST-30 kDa 4.1 was expressed
and purified in E. coli. By means of a His tag pull-down
assay, where purified His-tagged cdb3 was allowed to associate with
GST-30 kDa 4.1 and the complex was pelleted with the help of
Ni2+ beads, the affinity of mutated cdb3 for 4.1 was
examined. As shown in Fig. 2C, the amount of bound protein
4.1 associated with mutant cdb3, as measured from the GST activity in
the pellet, was the same as the quantity of protein 4.1 associating
with wild type cdb3, confirming that deletion of the
-hairpin loop
induces no global change in cdb3 conformation. Further,
glyceraldehydes-3-phosphate dehydrogenase, a glycolytic enzyme that
binds to cdb3 and becomes inhibited upon binding, was also examined for
any change in interaction with the mutant cdb3. As demonstrated in the
customary glyceraldehyde-3-phosphate dehydrogenase inhibition assay
(33), both wild type and mutant cdb3 were able to inhibit the enzyme
>90% with the same inhibition constant (data not shown). Taken
together, we conclude that any impact of loop deletion on ankyrin
binding must be attributed to a direct modification of the attachment
site of ankyrin and not to a general perturbation of cdb3 structure.
Loop Deletion
Mutant of cdb3--
Binding of ankyrin to wild type and mutant cdb3
was also evaluated by a His tag pull-down assay similar to that used
for analysis of protein 4.1 binding. Thus, His-tagged cdb3 was
incubated with a fusion construct of GST linked to the D3/D4 domains of
the membrane-binding domain of ankyrin (a construct of ankyrin
comprising ankyrin repeats 13-24 that has been frequently used to
study ankyrin-band 3 interactions (29, 34-36)) and any complexes
formed were pelleted by collection of the His-tagged cdb3 on
Ni2+ beads. The content of pelleted GST-46.5 kDa ankyrin
was then quantitated by measuring GST activity in the pellet. As shown in Fig. 3A, the binding
curve for the association of the GST-ankyrin fusion
construct with His-tagged cdb3 shows saturation with an apparent
dissociation constant of ~400 nM. Binding was found to be
specific because addition of excess unlabeled ankyrin competitively blocked the interaction and because heat denaturation of the 46.5-kDa ankyrin eliminated all association (data not shown). Importantly, binding of the 175-185 deletion mutant to ankyrin was not
distinguishable from background, suggesting that the deleted loop is
critical for ankyrin association. Further, when the same analysis was
performed by a dot-blot assay using His-tagged cdb3 and unmodified 46.5 kDa ankyrin, a qualitatively similar result was obtained (Fig. 3B). These data suggest that the
-hairpin loop on cdb3
plays a critical role in the ankyrin interaction.
View larger version (31K):
[in a new window]
Fig. 3.
A, comparison of the binding of the
46.5-kDa band 3-binding domain of ankyrin with purified recombinant
cdb3 and various mutant cdb3s. His-tagged wild type or mutant cdb3 was
incubated with increasing concentrations of GST-46.5 kDa ankyrin
overnight at 4 °C in binding buffer. Binding of GST-ankyrin was then
assayed by measuring GST activity in the pellet, as described in the
legend to Fig. 2C. B, dot blot analysis of 46.5 kDa ankyrin binding to purified wild type and mutant cdb3. Ankyrin-cdb3
complexes were formed as described above, eluted from the Ni-NTA
agarose beads with imidazole, and dot blotted onto a nitrocellulose
membrane. The membrane was then probed with anti-ankyrin antibody and
developed using goat anti-rabbit antibody coupled to horseradish
peroxidase plus a chemiluminescent substrate.
-hairpin loop for binding
than 46.5 kDa ankyrin, which lacks the first half of its
membrane-binding domain. To address this issue, we purified intact
ankyrin from the red cell membrane and radiolabelled it with
125I-Bolton-Hunter reagent. Following incubation with
His-tagged cdb3, the resulting ankyrin-cdb3 complexes were pulled-down
using Ni2+ beads, and bound ankyrin was quantitated by
measuring cpm. As seen in Fig.
4, intact ankyrin was also inhibited in
its association with the cdb3 deletion mutant, suggesting that even the
intact protein, with all 24 ankyrin repeats, relies heavily on the
-hairpin loop for interaction.
View larger version (15K):
[in a new window]
Fig. 4.
Evaluation of the binding of intact ankyrin
to purified recombinant cdb3 and mutant cdb3s. Intact ankyrin was
purified and labeled with 125I-Bolton-Hunter reagent and
allowed to associate with His-tagged cdb3. After collection of the
complexes on Ni-NTA agarose beads, ankyrin binding was quantitated by
measuring the radioactivity in the pellet.
-hairpin loop in situ. The resulting
looped peptide was then tested for its ability to inhibit binding of
46.5 kDa ankyrin to cdb3. As seen in Fig. 5, the synthetic peptide was indeed able
to completely inhibit ankyrin binding at concentrations around 500 µg/ml. Curiously, rupture of the disulfide bond using a reducing
agent did not attenuate the ability of the peptide to inhibit cdb3
binding (data not shown), suggesting that the cyclized peptide was not
highly constrained in its conformational flexibility.
View larger version (10K):
[in a new window]
Fig. 5.
Competitive inhibition of
GST-46.5 kDa ankyrin binding to wild type cdb3 by the
peptide comprising residues 175-185 of cdb3. Association of
GST-ankyrin with His-tagged cdb3 was measured (see methods in the
legend to Fig. 3A) in the presence of increasing
concentrations of either cyclized or reduced synthetic peptide
comprising residues 175-185 of cdb3. To enable cyclization, the
sequence (PAVLTRSGDPS) was modified by attaching cysteine residues at
each end.
-pleated sheet, which must
certainly perturb the global conformation of cdb3. Therefore, to better
evaluate the proposed involvement of the NH2 terminus of
cdb3 in ankyrin binding, a new deletion mutant was required that would
induce no distortion in cdb3 conformation. For this purpose, the
crystal structure of cdb3 was again consulted and interpreted to
indicate that the NH2-terminal 50 residues of cdb3 are
unstructured and can be eliminated without perturbing any other regions
of the polypeptide (25). Therefore, residues 1-50 of cdb3 were
deleted, and the resulting truncated cdb3 was evaluated for ankyrin affinity.
-hairpin loop sterically
obstructs phosphorylation of cdb3 on Tyr-8. Examination of the crystal
structure of cdb3 demonstrates that this steric overlap is entirely reasonable.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hairpin loop comprising
residues 175-185 of cdb3 constitutes a major ankyrin-binding site on
the erythrocyte membrane. Although previous studies (19-22) have suggested the involvement of a mid-region of cdb3 in ankyrin binding, a more precise characterization of this binding site could not
be pursued until the three-dimensional structure of cdb3 became
available. Thus, random deletion mutagenesis of the implicated
sequences without the aid of crystallographic coordinates would have
risked inducing global changes in protein conformation that might have
led to nonspecific inhibition of ankyrin association. With access to
the crystal structure, however, it was possible to identify and delete
the crucial loop region on cdb3 without introducing distortion or
strain into the rest of the protein. Abrogation of ankyrin binding by
this deletion mutant, with no accompanying perturbation of either the
pH-dependent conformational change or binding affinity for
protein 4.1, could then be interpreted to indicate the critical
involvement of the
-hairpin loop in ankyrin binding. This conclusion
was further confirmed by the ability of the deleted peptide to directly
compete for ankyrin association with cdb3.
-hairpin loop
inhibits phosphorylation of Tyr-8 argues strongly that the NH2 terminus and the ankyrin-binding site lie near each
other. Curiously, prediction of this steric interference would not have been obvious from the crystal structure because the first 55 residues of cdb3 are too flexible to be resolved and because the
-hairpin loop lies ~50 Å from residue 55 (i.e. the site where the
flexible NH2-terminal sequence leaves the body of cdb3).
Nevertheless, a close physical proximity must be real because ankyrin
also competes with other peripheral proteins that bind at the
NH2 terminus of band 3 (38)2. Because the
NH2 terminus of cdb3 is not only required for stable interaction with protein 4.1 (38) but also for association with various
glycolytic enzymes (33, 39) and p72syk (40, 41), it is also
conceivable that binding of ankyrin to cdb3 might be involved in
regulation of other protein interactions at the NH2 terminus.
sheet that extends through the middle of cdb3.
Although deletion of residues 1-50 can be argued to have little or no
impact on protein conformation, removal of residues 1-65 should force
rearrangement of the packing of the entire domain. Thus, based on
crystallographic considerations, kidney cdb3 should have a somewhat
different conformation from erythrocyte cdb3. Consistent with this
speculation, it has been reported that kanadaptin interacts with
kidney, but not erythrocyte band 3 (42), whereas the glycolytic
enzymes, ankyrin, and protein 4.1 associate with erythrocyte but not
kidney band 3 (24).
-hairpin loop, but neither preparation appears to involve docking with the NH2 terminus, even
though both are inhibited by Fabs directed at this sequence, and both prevent phosphorylation of Tyr-8. Previous studies (35) have suggested that ankyrin contains one cdb3-binding site on its second set
of 6 ankyrin repeats (subdomain 2) and another binding site somewhere
within repeats 13-24 (subdomains 3 and 4, i.e. the 46.5-kDa ankyrin. Based on the above similarities in binding properties, it is
tempting to speculate that most of the interfacial contacts may occur
between subdomains 3 and 4 and cdb3 and that docking with subdomain 2 may involve a more limited surface area.
-hairpin loop
constitutes a common ankyrin-binding motif on the cytoplasmic domains
of multiple membrane-spanning proteins. Even though the loop on cdb3
shares no sequence homology with the loop on the
Na+/K+-ATPase, cdb3 still inhibits the binding
of ankyrin to the Na+/K+-ATPase (43). We
interpret this to suggest that a conformational fit rather than
sequence complementarity at the binding interface contributes primarily
to complex formation. Indeed, our inability to compromise
ankyrin-binding affinity by mutating both charged residues in the
-hairpin loop (data not shown) is consistent with the more dominant
role played by conformational fit in stabilizing this interaction. As
other ankyrin-binding structures become available, it will be
interesting to examine whether similar exposed loops constitute the
sites of association.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Marko Stefanovic for providing recombinant 46.5 kDa ankyrin.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM24417.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.
To whom correspondence should be addressed. Tel.: 765-494-5273;
Fax: 765-494-0239; E-mail: plow@purdue.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211137200
2 S. H. Chang and P. S. Low, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: cdb3, cytoplasmic domain of erythrocyte membrane band 3; Ni-NTA, nickel-nitrilotriacetic acid; GST, glutathione S-transferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bennett, V., and Stenbuck, P. J. (1979) Nature 280, 468-473[Medline] [Order article via Infotrieve] |
2. | Koob, R., Zimmermann, M., Schoner, W., and Drenchhahn, D. (1988) Eur. J. Cell Biol. 45, 230-237[Medline] [Order article via Infotrieve] |
3. | Nelson, W. J., and Veshnock, P. J. (1987) Nature 328, 533-536[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Malhotra, J. D.,
Kazen-Gillespie, K.,
Hortsch, M.,
and Isom, L. L.
(2000)
J. Biol. Chem.
275,
11383-11388 |
5. |
Li, Z. P.,
Burke, E. P.,
Frank, J. S.,
Bennett, V.,
and Philipson, K. D.
(1993)
J. Biol. Chem.
268,
11489-11491 |
6. | Kalomiris, E. L., and Bourguignon, L. Y. (1988) J. Cell Biol. 106, 319-327[Abstract] |
7. |
Davis, J. Q.,
and Bennett, V.
(1994)
J. Biol. Chem.
269,
27163-27166 |
8. | Dubreuil, R. R., Macvicar, G., Dissanayake, S., Liu, C., Homer, D., and Hortsch, M. (1998) J. Cell Biol. 133, 647-655[Abstract] |
9. |
Bourguignon, L. Y.,
Jin, H.,
Iida, N.,
Brandt, N. R.,
and Zhang, S. H.
(1993)
J. Biol. Chem.
268,
7290-7297 |
10. |
Bourguignon, L. Y.,
Chu, A.,
Jin, H.,
and Brandt, N. R.
(1995)
J. Biol. Chem.
270,
17917-17922 |
11. | Lux, S. E., John, K. M, and Bennett, V. (1990) Nature 344, 36-42[CrossRef][Medline] [Order article via Infotrieve] |
12. | Bork, P. (1993) Proteins 17, 363-374[Medline] [Order article via Infotrieve] |
13. | Sedgwick, S. G., and Smerdon, S. J. (1999) Trends Biochem. Sci. 24, 311-316[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Michaely, P.,
and Bennett, V.
(1993)
J. Biol. Chem.
268,
22703-22709 |
15. | Devarajan, P., Scaramuzzino, D. A., and Morrow, J. S. (1994) Proc. Natl. Aacd. Sci. U. S. A. 91, 2965-2969[Abstract] |
16. | Srinivasan, Y., Lewallen, M., and Angelides, K. J. (1992) J. Biol. Chem. 276, 7483-7489 |
17. |
Bennett, V.,
and Baines, A. J.
(2001)
Physiol. Rev.
81,
1353-1391 |
18. |
Zhang, X.,
Davis, J. Q.,
Carpenter, S.,
and Bennett, V.
(1998)
J. Biol. Chem.
273,
30785-30794 |
19. |
Thevinin, B. J.-M.,
Willardson, B. M.,
and Low, P. S.
(1989)
J. Biol. Chem.
264,
15886-15892 |
20. |
Willardson, B. M.,
Thevenin, B. J.-M.,
Harrison, M. L.,
Kuster, W. M.,
Benson, M. D.,
and Low, P. S.
(1989)
J. Biol. Chem.
264,
15893-15899 |
21. |
Davis, L.,
Lux, S. E.,
and Bennett, V.
(1989)
J. Biol. Chem.
264,
9665-9672 |
22. |
Ding, Y.,
Kobayashi, S.,
and Kopito, R.
(1996)
J. Biol. Chem.
271,
22494-22498 |
23. |
Ding, Y.,
Casey, J. R.,
and Kopito, R.
(1994)
J. Biol. Chem.
269,
32201-32208 |
24. |
Wang, C. C.,
Moriyama, R.,
Lombardo, C. R.,
and Low, P. S.
(1995)
J. Biol. Chem.
270,
17892-17897 |
25. |
Zhang, D.,
Kiyatkin, A.,
Bolin, J. T.,
and Low, P. S.
(2000)
Blood
96,
2925-2933 |
26. | Zhang, Z., Devarajan, P., Dorfman, A. L., and Morrow, J. S. (1998) J. Biol. Chem. 1998, 18681-18684[CrossRef] |
27. |
Wang, C. C.,
Badylak, J. A.,
Lux, S. E.,
Moriyama, R.,
Dixon, J. E.,
and Low, P. S.
(1992)
Protein Sci.
1,
1206-1214 |
28. | Bennett, V. (1983) Methods Enzymol. 96, 313-324[Medline] [Order article via Infotrieve] |
29. |
Davis, L.,
and Bennett, V.
(1990)
J. Biol. Chem.
265,
10589-10596 |
30. | Low, P. S., Westfall, M. A., Allen, D. P., and Appell, K. C. (1984) J. Biol. Chem. 269, 13070-13076 |
31. |
Appell, K. C.,
and Low, P. S.
(1981)
J. Biol. Chem.
256,
11104-11111 |
32. |
Habig, W. H.,
Pabst, M. J.,
and Jakoby, W. B.
(1974)
J. Biol. Chem.
249,
7130-7139 |
33. | Tsai, L. H., Murthy, S. N. P., and Steck, T. L. (1982) J. Biol. Chem. 157, 1438-1442 |
34. |
van Dort, H. M.,
Moriyama, R.,
and Low, P. S.
(1998)
J. Biol. Chem.
273,
14819-14826 |
35. |
Michaely, P.,
and Bennett, V.
(1995)
J. Biol. Chem.
270,
22050-22057 |
36. | van Dort, H. M., Knowles, D. W., Chasis, J. A., Lee, G., Mohandas, N., and Low, P. S. (2001) J. Biol. Chem. 276 |
37. | Lux, S. E., and Palek, J. (1995) in Blood: Principles and Practice of Hematology (Handinm, R. I. , Lux, S. E. , and Stossel, T. P., eds) , pp. 1701-1818, Lippincott, Philadelphia |
38. |
Lombardo, C. R.,
Willardson, B. M.,
and Low, P. S.
(1992)
J. Biol. Chem.
267,
9540-9546 |
39. |
Murthy, P. S. N.,
Liu, T.,
Kaul, R. K.,
Kohler, H.,
and Steck, T. L.
(1981)
J. Biol. Chem.
256,
11203-11208 |
40. |
Harrison, M. L.,
Isaacson, C. C.,
Burg, D. L.,
Geahlen, R. L.,
and Low, P. S.
(1994)
J. Biol. Chem.
269,
955-959 |
41. | Yannoukakos, D., Vasseur, C., Piau, J.-P., Wajcman, H., and Bursaux, E. (1991) Biochim. Biophys. Acta 1061, 253-266[Medline] [Order article via Infotrieve] |
42. |
Chen, J.,
Vijayakumar, S., Li, X.,
and Al-Awqati, Q.
(1998)
J. Biol. Chem.
273,
1038-1043 |
43. | Morrow, J. S., Cianci, C., Ardito, T., Mann, A., and Kashgarian, M. T. (1989) J. Cell Biol. 108, 455-465[Abstract] |