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Plenary paper
Crystallographic structure and functional interpretation of the cytoplasmic domain
of erythrocyte membrane band 3
Dachuan Zhang, Anatoly Kiyatkin, Jeffrey T. Bolin, and Philip S. Low
The red blood cell membrane (RBCM) is a
primary model for animal cell plasma
membranes. One of its major organizing
centers is the cytoplasmic domain of band
3 (cdb3), which links multiple proteins to
the membrane. Included among its peripheral protein ligands are ankyrin (the major
bridge to the spectrin-actin skeleton), protein 4.1, protein 4.2, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phos-
phofructokinase, deoxyhemoglobin, p72syk
protein tyrosine kinase, and hemichromes.
The crystal structure of cdb3 is reported at
0.26 nm (2.6 Å) resolution. A tight symmetric dimer is formed by cdb3; it is stabilized by interlocked dimerization arms
contributed by both monomers. Each subunit also includes a larger peripheral protein binding domain with an ␣ⴙ ␤-fold.
The binding sites of several peripheral
proteins are localized in the structure,
and the nature of the major conformational change that regulates membraneskeletal interactions is evaluated. An improved structural definition of the protein
network at the inner surface of the RBCM
is now possible. (Blood. 2000;96:
2925-2933)
© 2000 by The American Society of Hematology
Introduction
The red blood cell membrane (RBCM) is constructed primarily of
an anastomosing spectrin-actin protein skeleton connected at
regular intervals to the integral membrane proteins, band 3 and
glycophorin C, via the bridging proteins, ankyrin, and protein 4.1.1
Because the membrane is easily isolated, architecturally simple,
and compositionally related to many other membranes, it is
commonly presented as a model of animal cell plasma membranes
in biochemistry textbooks.2,3 However, although each of the major
membrane proteins has been scrutinized functionally, high resolution crystallographic data are available only for actin and a single
domain of spectrin.4-6
Band 3 constitutes the most abundant polypeptide in the
RBCM, comprising 25% of the total membrane protein. Band 3,
also termed the anion exchanger (AE1), can be cleaved into 2
independent structural domains that retain the functional characteristics of their counterparts in the intact protein. The 55-kd
membrane-spanning domain is thought to traverse the bilayer 12
times and serves to catalyze the exchange of anions (mainly Cl⫺ for
HCO3⫺) across the membrane during gas transport in the blood.7
The membrane-spanning domain may also mediate removal of
senescent RBCs from circulation,8-10 and it has been shown to carry
a number of common blood group antigens.11 A low (2.0 nm [20
Å]) resolution structure of the membrane-spanning domain has
been reported.12
The cytoplasmic domain of band 3 (cdb3) functions primarily
as an anchoring site for other membrane-associated proteins.
Included among the protein ligands of cdb3 are ankyrin,13 protein
4.2,14,15 protein 4.1,16,17 glyceraldehyde-3-phosphate dehydrogenase (GAPDH),18 phosphofructokinase,19 aldolase,20 hemoglobin,21,22 hemichromes,23 and the protein tyrosine kinase (p72syk).24
From the Departments of Chemistry and Biological Sciences, Purdue
University, West Lafayette, IN.
Supported by grant GM24417 from the National Institutes of Health (NIH),
Bethesda, MD. Use of the Advanced Photon Source was supported by contract
W-31-109-Eng-38 from the U.S. Department of Energy, Washington, DC. Use
of BioCARS Sector 14 was supported by grant RR-07707 from the NIH,
National Center for Research Resources, Bethesda, MD.
Submitted March 13, 2000; accepted June 23, 2000.
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
Importantly, each of these band 3 interactions has nontrivial
consequences for the structure and function of the cell, ranging
from control of cell flexibility and shape25 to regulation of glucose
metabolism,26 ion transport,27 and cell lifespan.10 Not surprisingly,
mutations mapped to band 3 have been reported to alter each of the
above characteristics including cell shape.27-33
Recognizing that cdb3 constitutes a major organizing center of
the RBCM and that structural information on cdb3 might allow a
more accurate model of the architecture and function of the
erythrocyte, we undertook to solve the crystal structure of cdb3 and
to re-evaluate the organization of the membrane in its vicinity. We
report here the structure of cdb3 at 0.26 nm (2.6Å) resolution.
Using previous information on the sites of cdb3 association with its
peripheral protein ligands, we also map these sites on cdb3 and
reconstruct a model of the membrane at this important locus.
Materials and methods
Experimental procedures
Residues 1-379 of the human erythrocyte band 3, encompassing the entire
sequence of cdb3 released by chymotryptic cleavage of erythrocyte
membranes, was expressed in Escherichia coli and purified to homogeneity,
as described elsewhere.34 X-ray diffraction patterns were measured from 1
selenomethionyl (SeMet)-substituted cdb3 crystal at 3 wavelengths near the
selenopotassium (SeK) absorption edge by the use of facilities associated
with BioCars beamline BM-14D (Advanced Photon Source, Argonne
National Laboratories, ). Diffraction patterns were measured by the
rotation/oscillation method over a total range of 360° at each wavelength.
The diffraction patterns were analyzed with the HKL programs35 and were
consistent with the space group and unit cell of the native crystal form
Reprints: Philip S. Low, Department of Chemistry, 1393 Brown Bldg, Purdue
University, West Lafayette, IN 47907-1393; e-mail: [email protected]., or
Jeffrey T. Bolin, Department of Biological Sciences, 1392 Lilly Hall, Purdue
University, West Lafayette, IN 47907-1392; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
2925
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2926
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
ZHANG et al
previously described.34 The crystal was flash frozen immediately prior to
exposure to x-rays and was maintained at a nominal temperature of 108 K
throughout the diffraction experiments. It had dimensions of
0.2 ⫻ 0.25 ⫻ 0.3 mm and diffracted to 0.26 nm (2.6 Å). Representative
statistics describing the diffraction data are presented in Table 1.
Initial phase estimates were obtained by the multiple-wavelength
anomalous diffraction method. The program SOLVE36 was used to establish
positional and scattering parameters for 16 Se sites within the asymmetric
unit and to calculate initial phase estimates. The CCP4 package37 was used
for most of the subsequent crystallographic calculations. Prior to model
building, the phases and electron density maps were improved through
density modification without averaging by use of the CCP4 program DM.38
The secondary structure was easily recognized in the maps. The backbone
structures of the 4 monomers were independently traced and then compared
to establish initial polyalanine models for each monomer. These models
were then independently elaborated to include side chains prior to
refinement.
The structure was refined by the use of XPLOR39 and O.40 Following
initial refinement by simulated annealing in the presence of noncrystallographic symmetry (NCS) restraints, the model was further refined
by restrained positional and temperature factor adjustments as well as
manual model rebuilding. The NCS restraints were not used in the latter
stages of refinement. The properties of the refined model are also provided
in Table 1.
Evaluation of the locations of the C-termini was problematic because
the 4 monomers in the asymmetric unit exhibited variably ordered
structures beyond residue 348. Although the P and S monomers are not well
defined beyond this point, the Q and R monomers have very similar,
well-defined backbone structures through residue 356, and the C-terminal
residues of monomer Q are not directly affected by crystal contacts.
Therefore, to compare the locations of the C-termini among the 3 possible
tetrameric complexes, monomer Q was superimposed on all monomers, and
the transformed locations of C␣:356Q were used as markers for the
C-termini. Similarly, the transformed location of C␣55Q was used as the
last resolvable N-terminal residue for each subunit.
Results
Crystallization and data collection
The cdb3 was crystallized at pH 4.8 by the sitting drop vapor
diffusion method followed by streak-seeding of the harvested
crystals into solutions of higher protein content, as described
previously.34 Crystals of the longest dimension (approximately 0.3
mm) that diffracted x-rays to resolutions up to approximately 0.21
nm (2.1Å) were obtained. Unfortunately, isomorphous heavy atom
derivatives using various reagents could not be prepared either due
to poor heavy atom substitution or because of an induced change in
crystal structure. Therefore, SeMet-substituted cdb3 was expressed
in an E coli host that was inhibited in endogenous methionine
biosynthesis,41 and the crystals were grown as described above.
X-ray diffraction analysis revealed a C2 space group with the
following unit cell dimensions: a, 18.159 nm (181.59 Å); b, 9.258
nm (92.58 Å); c, 12.333 nm (123.33 Å); and ␤, 131.86°. The
asymmetric unit includes the equivalent of 4 cdb3 monomers. The
current structure consists of 9509 nonhydrogen protein atoms and
178 water molecules and was refined to 0.26 nm (2.6 Å) resolution
and an R factor of 22% (Rfree ⫽ 29%). Properties of the refined
model are provided in Table 1.
Structure of cdb3 monomer
The cdb3 monomer includes 11 ␤-strands and 10 helical segments
and belongs to the ␣ ⫹ ␤–fold class (Figure 1A). Eight of the
␤-strands are assembled into a central ␤-sheet of mixed parallel and
antiparallel strands (Figure 1B). Two of the remaining strands (␤6
and ␤7, residues 176-185) form a ␤-hairpin that likely constitutes
the core of the ankyrin binding site, as described below. Along with
the first 6 helices, these elements assemble into a globular domain
spanning residues 55-290. A short helix and loop segment, residues
291-303, connects this large domain to a dimerization arm, which
comprises residues 304-357. This arm is largely helical, but it
includes a single ␤-strand, which plays a critical role in the
formation of an interlocked dimer (Figure 2A). The monomer is
compact, having dimensions of approximately 4.3 ⫻ 4.5 ⫻ 5.3 nm
(43 ⫻ 45 ⫻ 53 Å). There are no major structural differences
among the 4 monomers in the asymmetric unit, although there are
variations in the regions of intermolecular contact and in the extent
of order in the N- and C-terminal segments.
Residues 1-54, 202-211, and 357-379 were not observed or
were poorly defined by the electron density. Flexibility was indeed
expected in the first 54 residues because this region is strongly
anionic, containing 20 acidic residues, no basic residues, and a
blocked N-terminal methionine.42,43 The region is also involved in
Table 1. Summary of diffraction data and selected refinement statistics
Diffraction measurements
Wavelength, nm (Å)
0.09789 (0.9789)
0.09796 (0.9796)
0.09537 (0.9537)
Resolution, nm (Å)
3.5-0.26 (35.0-2.6)
3.5-0.26 (35.0-2.6)
3.5-0.26 (35.0-2.6)
0.057 (0.208)
0.049 (0.192)
0.052 (0.175)
99.1 (94.5)
98.2 (92.9)
97.6 (56.1)
0.216 (41 136)
0.290 (3 612)
Rsym, % (last shell, %)*
Completeness, % (last shell, %)
Current model and refinement statistics
R, Rfree (reflections)†
Atoms, no.
Nonhydrogen
9 687
Protein
9 509
Water
178
R.m.s. deviations‡
Bonds, nm (Å)
Angles, deg
0.00080 (0.0080)
1.67
Deg indicates degrees.
*Rsym indicates ␴IHKL ⫺ ⬍ IHKL ⬎ ␴IHKL, where IHKL is the intensity for one observation of a reflection and ⬍ ⬎ is the mean for all observations of that reflection.
†R indicates ␴Fobs ⫺ Fcal/␴Fobs, where Fobs and Fcal are the observed and calculated structure factor amplitudes, respectively. Rfree was calculated using the same formula
as Rcrystal, but with only 8% of the total amplitudes chosen randomly.
‡R.m.s. deviation is the root-mean-square deviation from the expected bond lengths or angles.
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
STRUCTURE OF THE CYTOPLASMIC DOMAIN OF BAND 3
2927
locations of the last resolvable residues at the N- and C-termini of
cdb3 are marked in Figures 1B and 2A. How these locations impact
the various peripheral protein interactions will be evaluated below.
Dimerization of cdb3
Isolated cdb3 has been shown to exist as a dimer over a range of
protein concentrations and solution conditions.51,52 This strong
preference for dimerization is readily explained by the crystal
structure. Within the asymmetric unit are 2 independent but
equivalent dimers, P-Q and R-S (Figure 3A), that are essentially
2-fold symmetric and interlocked by extensive interactions within
Figure 1. Structure of cdb3 monomer. (A) Diagram of cdb3 secondary structure with
the following sequence assignments: ␤157-66, ␤273-82, ␤384-88, ␤492-96, ␣1104-116,
␤5117-122, ␣2128-141, ␣3146-158, ␣4166-170, ␤6176-180, ␤7182-185, ␣5196-201, ␣6212-220,
␤8224-234, ␤9239-247, ␤10263-271, ␣7278-290, ␣8292-300, ␣9304-316, ␤11318-323, and ␣10328-347.
(B) Ribbon diagram of cdb3 monomer, with blue corresponding to the peripheral
protein binding domain and red to the dimerization arm. (C) Stereo drawing of the
␣-carbon trace for the cdb3 monomer in approximately the same orientation as in
panel B. Colors change from blue at the N-terminus to red at the C-terminus.
most peripheral protein interactions,44 suggesting that its conformation may be adaptable to a variety of protein ligands. The
N-terminal segment has, in fact, been shown by solution NMR and
x-ray crystallography to bind aldolase45 and deoxyhemoglobin21 in
different extended conformations. The inability to resolve residues
202-211 and 357-379 is also assumed to be due to segmental
flexibility. Physical studies of cdb3 have shown the protein to be a
highly flexible molecule.46,47
The positions of the N- and C-terminal segments are important
in defining the interactions of cdb3. The C-terminal residues of
cdb3 connect to the membrane-spanning domain, and hence, must
orient toward the lipid bilayer. The N-terminal segment, in contrast,
must interact with peripheral proteins, eg, the glycolytic enzymes,
hemoglobin, protein 4.1, ankyrin, and hemichromes.21,23,48-50 To
bind these proteins without steric interference from the bilayer, the
N-termini must presumably face away from the membrane. The
Figure 2. Structure of cdb3 dimer. (A) Ribbon diagram of cdb3 dimer showing
interactions between interlocking dimerization arms. The positions of the 2 cysteines
(Cys 201A and Cys 317B) that form an inter-subunit disulfide bond upon treatment
with the oxidizing agent, o-phenanthroline/CuSO4 (pH at least 7.5), are also shown.
Because these cysteines cannot form a disulfide in the low pH conformation of cdb3,
they are not expected to reside near each other in the low pH structure shown here.
(B) Space filling model of cdb3 dimer. (C) Cluster of 9 leucine residues (extending
from ␤11 and ␣10 of each subunit) that stabilize the dimerization domain.
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2928
ZHANG et al
Figure 3. Structure of cdb3 tetramer. (A) The packing of selected cdb3 dimers in
the crystal illustrated schematically by projection onto an a, c plane of the unit cell.
The unit cell includes 4 monomers of types P, Q, R, and S. (B) The association of cdb3
subunits in the likely physiological tetramer, with each potential ankyrin binding loop
(residues 176-190) highlighted in red. The plane of the bilayer is believed to lie in a
horizontal plane at the top of the diagram. (C) Relative orientation of the truncated
N- and C-termini of the tetramer, as viewed from one end of the tetramer. Color coding
is the same as in Figure 3B. Panels A and B are rotated with respect to each other by
180° about a horizontal axis in the plane of the page.
an intermonomeric dimerization domain (Figure 2A). To build this
domain each monomer contributes a dimerization arm (residues
314-344) that extends away from the major globular domain and
interdigitates with the equivalent arm from the second monomer
(Figure 2B). Among the extensive interactions at this interface are
8 intermonomeric backbone-to-backbone hydrogen bonds contributed in part by the 2 strands of the intermonomeric antiparallel
␤-sheet. Subunit association is further stabilized by a hydrophobic
core of 9 interacting leucine residues (Figure 2C). Approximately
520 nm (5200 Å)2 of exposed surface is buried at the dimer
interface, and 370 nm (3700 Å)2 of the buried surface is associated
with nonpolar side chain atoms. The 2 markers for the C-termini lie
near the center of the same face of the dimer, where they could
easily attach to adjacent membrane-spanning domains. The 2
markers for the N-termini locate on the lateral surfaces of the
dimer, where they would allow extension of the nonresolved
N-terminal residues away from the membrane. Figure 2B also
shows the general shape of the dimer, which fits within a
rectangular prism of approximate dimensions 7.5 ⫻ 5.5 ⫻ 4.5 nm
(75 ⫻ 55 ⫻ 45 Å).
Conformational equilibrium in cdb3 expansion of the dimer
at high pH
A remarkable property of cdb3 is its reversible, pH-dependent
conformational equilibrium, which is readily detected by intrinsic
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
fluorescence, sedimentation analysis, differential scanning calorimetry, fluorescence energy transfer, gel filtration chromatography,
native gel electrophoresis, and a variety of functional assays.44,46,47,52,53 Thus, as the pH is raised from 6.5 to 9.5, the Stokes
radius of cdb3 enlarges reversibly by 1.1 nm (11 Å), and the
intrinsic fluorescence, which is highly quenched at a lower pH,
more than doubles. A disulfide bond that can form between cysteine
(Cys) 201 of one subunit and Cys 317 of the paired subunit at a pH
of at least 7.5 can no longer be generated below pH 7.0, as seen in
locations of participating cysteines in low pH conformation in
Figure 2A, and cdb3-associated hemoglobin pivots away from the
membrane.53 Because ankyrin cannot bind to the high pH form, but
avidly associates with the low pH form,25,54 the nature of this
conformational equilibrium is important to understanding the
regulation of membrane-skeleton interactions.
We have examined the structure of cdb3 for a possible hinge
around which adjacent regions of cdb3 might pivot, thereby
generating the conformational change. Both the peripheral protein
binding domain and dimerization arm are tightly folded structures,
and therefore, we doubt that the conformational change can arise
solely within one of these regions. Two short segments between
these domains, however, could accommodate the anticipated
bending. The first, between helices ␣7 and ␣8, is centered at
glutamic acid (Glu) 291, and the second, between ␣8 and ␣9, is
centered at glutamine (Gln) 302. A change in the backbone
conformation at either or both locations would permit considerable
expansion of cdb3 while leaving the dimer tightly coupled through
the unmodified dimerization domain. Such a pivoting motion
would also explain why the aforementioned disulfide bond cannot
form below pH 7.0 and why its formation at pH 8.0 prevents
ankyrin association.54
Although crystallographic data were not available at a high pH,
we rotated the respective domains between helices ␣8 and ␣9 (at
glycine [Gly] 305) to visualize a possible high pH conformation of
the dimer. As seen in Figure 4, this operation not only elongated the
dimer, but it also disjoined a hydrogen bond connecting tryptophan
(Trp) 105 of one subunit to aspartic acid (Asp) 316 of the other.
Because this H bond would be expected to quench the Trp
fluorescence,55 this structural change could account for both the
increase in Trp fluorescence and cdb3 elongation.47 The decrease in
thermostability of the high pH form might also be explained by
disruption of this and other interactions present in the more compact
Figure 4. Possible expansion of cdb3 dimer at elevated pH. To envision a
possible high pH conformation of cdb3, the 2 peripheral protein binding domains were
rotated away from their shared dimerization domain by allowing them to pivot at Gly
305 (indicated by arrows), a plausible site for rotation to occur. The resulting protein
would be expected to remain dimeric, but also to become more elongated and
fluorescent as the domains separate and the quenching H bonds between Asp 316
and Trp 105 (shown in red) disjoin.
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
(low pH) structure. The decline in cooperativity of thermal denaturation with increasing pH might further derive from the separation
of tightly coupled domains into noninteracting domains.52
The cdb3 tetramer
In its membrane environment, intact band 3 exists primarily as a
mixture of dimers and tetramers.56-59 Because cdb3 is thought to be
essential for tetramer formation60-62 and because tetramer formation is critical for ankyrin association,54,56,63-65 we examined the
interactions within the crystal for contacts that might correspond to
the tetramer interface in vivo. The crystal structure revealed 3
possible tetramers defined by association of different R-S dimers
with a central P-Q dimer (Figure 3A). These are R2-S2:P-Q or
P-Q:R3-S3 (tetramer I), P-Q:R-S (tetramer II), and P-Q:R1-S1
(tetramer III). Remarkably, 2 of the tetramers (I and II) are
approximately 2-fold symmetric, and the third (tetramer III)
involves a 2-fold rotation coupled to a moderate translation.
Although identification of the physiological tetramer cannot
be established from crystallographic data, 4 lines of evidence
favor tetramer I as the likely native oligomer. First, locations of
contacts among subunits in tetramers II and III prohibit bending
and/or pivoting of the cdb3 dimerization arm relative to its
peripheral protein binding domain, thereby blocking the likely
conformational change in cdb3 seen both in situ and in vitro.44
This restriction is not seen in tetramer I. Second, the closed
tetramers in models II and III cannot elongate into hexamers and
higher oligomers, even though such extended forms of band 3
have been observed.29,51,63,66-68 Tetramer I, in contrast, is capable
of head-to-tail extension. Third, binding of ankyrin to its likely
site at residues 176-190 would obstruct the interfacial contacts
in tetramers II and III (eg, residues 187-190), thereby preventing
rather than promoting the ankyrin-induced tetramer formation
seen in situ.56,69,70 No such obstruction exists in tetramer I. And
finally, the noncontiguous regions of cdb3 involved in ankyrin
binding are proximal in tetramer I but further apart in tetramers
II and III, as indicated below.
Tetramer I is formed by 2 crystallographically independent but
structurally identical associations of P-Q and R-S dimers, ie,
R2-S2:P-Q and P-Q:R3-S3 (Figure 3A). In projection the tetramer
is approximately 14.0 nm (140 Å) in the longest dimension and 4.5
nm (45 Å) in the cross-section. Only 2 monomers, S with P or Q
with R, are in contact at the dimer:dimer interface, and the amino
acids in contact comprise residues 208-225 and 252-261 from both
subunits. Approximately 200 nm (2000 Å)2 of exposed surface is
buried at this interface, and 170 nm (1700 Å)2 is associated with
nonpolar side chain atoms.
Figure 3B shows the shape and subunit orientations in the above
tetramer, and Figures 3B and 3C indicate the relative locations of
the last resolvable residues at the N- and C-termini of each
polypeptide. The C-terminal markers (locations of serine [Ser] 356)
project onto an arc 130° wide and are thus approximately on the
same face of cdb3, thereby allowing simultaneous communication
with the membrane-spanning domains via the unresolved sequences, residues 357-399 (Figure 3B). Two of the N-terminal
markers (locations of histidine [His] 55) also extend from a face of
the tetramer that orients toward the membrane, whereas the other 2
are on lateral surfaces and fully accessible. Although this tetramer
is consistent with much published data, it will still be important
to verify its organization through biochemical studies in the
near future.
STRUCTURE OF THE CYTOPLASMIC DOMAIN OF BAND 3
2929
Ankyrin association with cdb3
Virtually all investigators agree that ankyrin binds primarily to the
tetramer of band 3, with little or no affinity for the dimer.54,55,6365,69,70 The addition of ankyrin to RBCM forces band 3 to a
tetramer.53,55,69,70 As a result, it is not unlikely that the ankyrin
binding site comprised sequences which were contributed by 2
separate dimers. The addition of ankyrin tethers these dimers
together, perhaps via association with 2 distinct band 3 binding
domains on the ankyrin.71 Importantly, the specific residues on
cdb3 involved in ankyrin binding have been partially mapped to at
least 2 noncontiguous regions. Antibody competition studies,
site-directed mutagenesis, chimera analysis, and phosphorylation
inhibition experiments all implicate the unresolved N-terminal
segment in this interaction.49,72-75 Similar methods plus proteolysis
protection studies further demonstrate involvement of residues
176-190, with additional contributions possible from adjacent
residues. As illustrated in Figures 1A and 3B, residues 176-190 lie
near the middle of a largely exposed and convoluted 100-residue
segment located between strands ␤5 and ␤8 of the main sheet.
Residues 177-185 include strands ␤6 and ␤7 and take the form of a
well-known ␤-hairpin structure.76 Evidence that this hairpin structure may indeed constitute an ankyrin binding site also stems from
recent crystallographic results which show that the putative ankyrin
docking segment from the sodium/potassium–adenosine 5⬘triphosphatase (Na⫹/K⫹-ATPase) also forms a stalk-and-loop structure when fused to the C-termini of glutathione S transferase.77
As noted above, an N-terminal segment must also contribute to
the high affinity binding site for ankyrin. In tetramer I the last
resolvable N-terminal amino acid from 1 dimer (His 55) lies in
reasonable proximity, 3.1-4.1 nm (31-41 Å), and on the same
surface of the tetramer as residues 176-190 from the other dimer
(Figure 3B). Not only does this attachment site orient ankyrin
toward the cytoplasm, where it can easily bridge to the spectrin
skeleton, but it also necessitates tetramer formation because
juxtaposition of an N-terminal segment and an ankyrin binding
loop requires the association of 2 dimers. In contrast, the distances
in tetramer II range from 5.9-7.6 nm (59-76 Å), and the 2 segments
implicated in ankyrin binding lie on different faces of the tetramer.
A possible arrangement of ankyrin on the extended band 3 tetramer
is depicted in Figure 5.
Association of cdb3 with other peripheral proteins
Protein 4.1 is the only peripheral protein known to associate
partially with residues in the dimerization arm of cdb3. In addition
to the required contact at the N-termini,50 protein 4.1 also binds to
an LRRRY sequence that forms part of a helix near the C-termini
(residues 343-34678; Figure 6). Based on biochemical studies, the
LRRRY sequence is believed to interact with an oppositely charged
LEEDY peptide in protein 4.1. However, because the side chains of
I343, R346, and Y347 are on less accessible surfaces of the helix,
any model that specifies direct pairing of IRRRY with LEEDY
would require either partial unfolding of the helix or only
delocalized interactions with the more buried residues. Importantly,
the marker for each subunit’s N-terminus lies sufficiently close to
its LRRRY sequence to allow binding of 4.1 to a single monomer.
Further, the corresponding sites on the 2 monomers of a cdb3 dimer
appear sufficiently separated to allow both monomers to bind a
different protein 4.1 simultaneously. While association of protein
4.1 with the extended cdb3 tetramer may also be possible, steric
hindrance from the membrane should afford preference to complexes with cdb3 N-termini pointing away from the bilayer. This
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
ZHANG et al
N-termini of the tetramer must exit the tightly folded core of the
oligomer on the membrane-apposed surface, at least a fraction of
the above enzyme binding sites can be anticipated to lie close to the
bilayer (Figure 5).
Noting that many associations involve the N-terminal segment
of cdb3, it might seem that competition for binding would be a
significant factor in regulating the assembly of complexes on cdb3.
However, there are sufficient molecules of band 3 in RBCs to bind
all copies of ankyrin, protein 4.1, and the aforementioned glycolytic enzymes simultaneously.44 Not all peripheral proteins can,
however, associate with the same oligomer of band 3 simultaneously (Figure 5).
The structure of kidney cdb3
Figure 5. Cartoon displaying the possible organization of a peripheral protein
complex on a cdb3 tetramer in situ. Protein 4.1 is not displayed in the diagram
because it competes with ankyrin for an overlapping site. Deoxy Hb indicates
deoxyhemoglobin; G3PD, glyceraldehyde-3-phosphate dehydrogenase.
would place protein 4.1 in direct competition with ankyrin for the
N-termini of subunits Q and R in the cdb3 tetramer. Competition
between protein 4.1 and ankyrin for cdb3 has, in fact, been reported
by several laboratories.16,50,79
Information on the binding site of protein 4.2 on cdb3 is limited.
Natural mutations at Glu 40, Gly 130, and proline (Pro) 327 of cdb3
lead to a protein 4.2 deficiency and hereditary spherocytosis.28,30,31
Unfortunately, none of these mutations colocalize to a common
region of cdb3. Pro 327 lies in the cdb3 dimerization domain and
hence its mutation to arginine (Arg) could impact self association.
Gly 130 is situated in an ␣-helix near the ankyrin binding loop and
its replacement with Arg could affect local folding. The Glu 40 to
lysine (Lys) charge reversal could compromise electrostatic interactions with the N-termini. Further studies will obviously be necessary to resolve which if any of these mutations identifies the
binding site of protein 4.2.
The glycolytic enzymes (GAPDH, aldolase, and phosphofructokinase), deoxyhemoglobin, and hemichromes all bind to the
extreme N-terminal segment of cdb3.21,23,48,80 These interactions,
which are largely electrostatic in nature, lead to inhibition of
glycolysis, reduced hemoglobin-oxygen affinity, and the clearance
of RBCs from circulation, respectively.8,21,26 Because p72syk also
associates with band 3 and phosphorylates tyrosine (Tyr) 8,24,81 it
may also interact within the same region. Given that 2 of the
Figure 6. Location of the protein 4.1 binding site on the cdb3 dimer. The side
chain orientations of the LRRRY sequence are shown, and the location of the
proximal N-terminal marker is labeled to allow visualization of the protein 4.1
binding surface.
The kidney band 3 is identical to the RBC band 3 except that
translation of the polypeptide in the kidney band 3 begins at exon 5,
resulting in the absence of residues 1-65. As seen in Figure 7, this
alternative initiation site also causes deletion of a central strand in
the major ␤-sheet. How the remaining folded structure of cdb3
accommodates removal of this middle strand is intriguing. However, it should not be overlooked that the functional differences
between erythrocyte and kidney cdb3 are not minor because kidney
cdb3 no longer binds ankyrin, protein 4.1, or the glycolytic
enzymes.74,75 Thus, the N-terminal 65 residues of cdb3 may be
critical to band 3–specific functions.
Discussion
The significance of the cdb3 structure and function to erythrocyte
properties is emphasized by the fact that missense mutations in
cdb3 can result in global changes in RBC shape and deformability.28,30-32 Although the deleterious effects of 1 or 2 of these amino
acid substitutions can possibly be explained by localization to the
binding site of an important peripheral protein,28,30 others appear
more likely to derive from their potential impact on cdb3 conformation. Indeed, 3 mutations with a significant effect on erythrocyte
morphology reside in conserved regions of the secondary structure
that are far removed from assumed docking sites of peripheral
Figure 7. Ribbon diagram of the cdb3 monomer. The dark strand in the middle is
the ␤-strand that is deleted from the kidney cdb3.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
proteins.32 Thus, band 3 Tuscaloosa (Pro 327 3 Arg), band 3
Boston (alanine [Ala] 285 3 Asp), and band 3 Nachod (GTVLL
117-121 deleted) all contain mutations that localize to helices or
sheets which may be critical to the correct folding of the
polypeptide (Figures 1 and 2). The effects of these mutations on
erythrocyte morphology would argue that the native conformation
of cdb3 is essential to the structure and function of the entire
erythrocyte.
In contrast to the above mutations, amino acid substitutions in
unstructured regions of cdb3 may not cause alteration in cell
morphology. An example of this class of polymorphism is found in
band 3 Memphis,82 where Lys 56 is replaced by Glu directly
upstream of the first strand of the central 8–stranded ␤-pleated
sheet. In this instance the substitution results in a 20% reduction in
the rate of anion transport across the membrane.82 From the
position of the Lys 3 Glu substitution near the attachment of cdb3
to the membrane-spanning domain, we speculate that the change in
electrostatic charge might reduce the concentration of anions near
their transport site at the membrane surface.
The erythrocyte band 3 has been sequenced from a variety of
organisms including human, cow, rat, mouse, chicken, and
trout.42,43,83-86 A comparison of these sequences reveals extended
regions of sequence homology interconnected by regions of
moderate divergence. When the regions of homology are aligned
with the 3-dimensional map of cdb3, it is observed that they
correspond with high fidelity to the secondary structural elements
displayed in Figure 1. This suggests that the general fold of cdb3
has been conserved among organisms examined to date, even
though the erythrocytes of birds and fish are nucleated and
stabilized by a classical actin- and tubulin-based cytoskeleton. Of
further relevance, the sequences conserved in AE2 and AE3 (ie,
other members of the anion exchanger gene family) also correspond to the above regions of secondary structure, which suggests a
related fold for these more divergent proteins. In fact, the sequences of greatest divergence among all isoforms of band 3
constitute loops of different lengths connecting the well-defined
segments of the protein secondary structure.
One of the more surprising features to emerge from the crystal
structure was the overall shape of cdb3. As noted above, the pH 4.8
crystal form of the cdb3 dimer has dimensions of approximately
7.5 ⫻ 5.5 ⫻ 4.5 nm (75 ⫻ 55 ⫻ 45 Å). In contrast, hydrodynamic
measurements of the solution form of the same dimer yield Stokes
radii estimated at 4.65 nm (46.5 Å; pH 8)51 to 5.3 nm (53 Å; pH
7.4).52 Further, sedimentation measurements at pH 8 yield an estimate
of P ⫽ 1.5 (approximately), which in turn predicts an axial ratio of
10:1 for an equivalent prolate ellipsoid of revolution.51 In contrast,
the atomic coordinates of the interlocked dimer yield a calculated
P ⫽ 1.04 (approximate) and an axial ratio of 1.9:1.
Various experimental factors could obviously account for these
discrepant results, but 3 explanations deserve further consideration.
First, the hydrodynamic estimates could be flawed. Although
reasonably similar Stokes radii have been reported by 2 independent labs,51,52 major assumptions were still required in these
measurements, and these assumptions could have lead to distorted
estimates. Second, the crystallized conformation of cdb3 could be
STRUCTURE OF THE CYTOPLASMIC DOMAIN OF BAND 3
2931
nonphysiological. However, while minor differences between cdb3
in situ and the crystallized structure are not unlikely, the ability of
the crystal structure to explain many previous functional and
structural observations argue against this interpretation; eg, the
strong preference of cdb3 to dimerize, the ability of protein 4.1 to
simultaneously dock at both the N- and C-termini of cdb3, the
presence of a bendable hinge between 2 independent domains in
cdb3, the inability of kidney cdb3 to bind any of erythrocyte cdb3
peripheral protein ligands, the tendency of ankyrin to force band 3
tetramer formation, and the nature of the conserved sequences in
cdb3. Still, one must recognize that cdb3 undergoes a reversible
structural change as the pH is lowered, becoming increasingly
more compact and thermally stable.44,46,47,52,53 So it is not unreasonable to expect some differences between the conformation described here and the predominant form at pH 7.4. Further studies
guided by the crystal data will obviously be required to resolve this
issue. Finally, neither the free N-termini nor free C-termini are seen
in the crystal structure. Thus, if the unresolved N-terminal 54 and
C-terminal 23 amino acids were to form highly extended structures,
the axial ratio derived from the hydrodynamic experiments would
be significantly greater than the dimensions measured in the
crystal. The observations that the N-terminus of cdb3 binds both
hemoglobin21 and aldolase45 in an extended conformation would
favor this interpretation.
With the tertiary structure of the low pH conformation of cdb3
now established, it should be possible to construct a more
predictive model of the architecture of the erythrocyte membrane.
It is already apparent that the site of the ankyrin attachment to the
membrane cannot be much more than approximately 6.0 nm (60 Å)
from the lipid surface87 because the cross-sectional dimension of
the cdb3 orthogonal to the membrane is only approximately 5.0 nm
(50 Å), not 25.0 nm (250 Å) as previously supposed.44 The position
of protein 4.1 binding is also more accurately defined because the
LRRRY sequence resides in the dimerization arm near the interface
between the subunits. Glycolytic enzymes, in contrast, may form a
ring around each cdb3 oligomer as they associate with available
radiating N-termini (Figure 5). It is even conceivable that an
enclosed metabolite compartment, such as that envisioned by
Hoffman,88 could assemble on the cdb3 oligomer if interactions
among the glycolytic enzymes were correctly arranged. Finally, the
crystal structure of cdb3 now provides a central framework on
which to build a more elaborate membrane model as further
peripheral protein structures are solved. In the absence of a
technology that allows resolution of complex noncrystallizable
structures, the assembly of an intact structure from its well-defined
parts may constitute the best approach available.
Acknowledgments
Christopher L. Colbert is thanked for his assistance in the
diffraction experiments. John E. Johnson (while at Purdue University, West Lafayette, IN) is thanked for his guidance in growing
cdb3 crystals and searching for heavy atom derivatives. Stephen
Hending is thanked for discussions of hydrodynamic measurements.
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2000 96: 2925-2933
Crystallographic structure and functional interpretation of the
cytoplasmic domain of erythrocyte membrane band 3
Dachuan Zhang, Anatoly Kiyatkin, Jeffrey T. Bolin and Philip S. Low
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