Localization of the ankyrin-binding site on erythrocyte membrane protein, band 3

The predominant attachment site of the spectrin-based cytoskeleton to the erythrocyte membrane occurs via the interaction of ankyrin with the cytoplasmic domain of band 3 (cdb3). In order to further characterize this interaction, we have conducted experiments to localize the ankyrin-binding site on cdb3. Four monoclonal and three antipeptide polyclonal antibodies were raised against cdb3 and used in competition studies to identify regions of close association of cdb3 with ankyrin. Antibodies to regions of cdb3 near the cytoplasmic domain-membrane spanning domain junction had no effect on 125I-ankyrin binding. Likewise, an antibody to a highly conserved region between residues 142 and 154 did not inhibit ankyrin binding. However, antibodies at or near the cysteine 201-317 cluster and the proposed proline-rich hinge in the center of cdb3 were potent inhibitors of ankyrin association, as were antibodies to the acidic NH2 terminus. Additional evidence for interaction of ankyrin with the NH2-terminal region of cdb3 was obtained by demonstrating the ability of ankyrin to inhibit tyrosine phosphorylation of cdb3 at its NH2 terminus by a purified calf thymus tyrosine kinase. These studies reveal two regions of cdb3, distant in primary sequence, which interact with ankyrin. A specific conformation of cdb3 may be required to permit these regions to simultaneously associate with ankyrin and allow binding to occur.     

Identification of a critical ankyrin-binding loop on the cytoplasmic domain of erythrocyte membrane band 3 by crystal structure analysis and site-directed mutagenesis

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, p72(syk). The crystallographic structure of the cdb3 dimer has revealed that residues 175-185 assume a beta-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 beta-hairpin turn indeed constitutes a major ankyrin-binding site on cdb3. Other biochemical studies suggest that ankyrin also docks at the NH(2) terminus of band 3. Thus, antibodies to the NH(2) terminus of cdb3 block ankyrin binding to the cdb3, and ankyrin binding to cdb3 prevents p72(syk) phosphorylation of cdb3 at its NH(2) terminus (predominantly at Tyr-8). However, a truncation mutant of cdb3 lacking the NH(2)-terminal 50 residues displays the same binding affinity as wild type cdb3. These data thus suggest that the NH(2) terminus of cdb3 is proximal to but not required for the cdb3-ankyrin interaction.     

Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.

The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.     

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

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, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.     

Rh-RhAG/ankyrin-R,a new interaction site between the membrane bilayer and the red cell skeleton,is impaired by Rh(null)-associated mutation

Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.     

Rate of rupture and reattachment of the band 3-ankyrin bridge on the human erythrocyte membrane

The principal bridge connecting the erythrocyte membrane to the spectrin-based skeleton is established by band 3 and ankyrin; mutations leading to reduced bridge formation or increased bridge rupture result in morphological and mechanical abnormalities. Because membrane mechanical properties are determined in part by the protein interactions that stabilize the membrane, we have evaluated the rates of rupture and reattachment of band 3-ankyrin bridges under both resting and mechanically stressed conditions. To accomplish this, we have examined the rate of ankyrin displacement from inside-out vesicles by the hexahistidine-tagged cytoplasmic domain of band 3, cdb3-(His)6 and the rate of substitution of cdb3-(His)6 into endogenous band 3-ankyrin bridges in resealed erythrocytes in the presence and absence of shear stress. We demonstrate that 1) exogenous cdb3-(His)6 displaces endogenous ankyrin from IOVs with a half-time and first order rate constant of 42 +/- 14 min and 0.017 +/- 0.0058 min(-1), respectively; 2) exogenous cdb3-(His)6 substitutes endogenous band 3 in its linkage to ankyrin in resealed cells with a half-time and first order rate constant of 12 +/- 3.6 min and 0.060 +/- 0.019 min(-1), respectively; 3) cdb3-(His)6-mediated rupture of the band 3-ankyrin bridge in resealed cells results in decreased membrane mechanical stability, decreased deformability, abnormal morphology, and spontaneous vesiculation of the cells; and 4) the above on/off rates are not significantly accelerated by mechanical shear stress. We conclude that the off rates of the band 3-ankyrin interaction are sufficiently slow to allow sustained erythrocyte deformation without loss of elasticity.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Identification of the binding interface involved in linkage of cytoskeletal protein 4.1 to the erythrocyte anion exchanger.

Linkages of the cytoskeleton to integral membrane proteins of the plasma membrane have been shown to be important for diverse cellular functions. The erythrocyte membrane provides the best studied example of how the spectrin-actin based membrane cytoskeleton is linked via two proteins, ankyrin and protein 4.1, to the anion exchanger (anion exchanger 1, AE1). Although these and other types of cytoskeleton-membrane connections have been well documented by in vitro binding studies it has not been possible to establish any of such interactions by defining the binding interface at the amino acid level. In the present study we have performed binding studies between protein 4.1 and AE1 using peptides and corresponding idiotypic and anti-idiotypic antibodies to show that arginine-rich clusters of the cytoplasmic domain of AE1 (IRRRY/LRRRY) serve as a major binding site for a motif with opposite charge and identical hydrophobicity present on the membrane-binding domain of protein 4.1 (LEEDY). Both motifs appear to be highly conserved during evolution and may also be involved in other types of cytoskeleton-membrane association, i.e. in binding of protein 4.1 to the glycophorins.     

利用核糖体展示技术筛选功能性蛋白质方法的建立

核糖体展示(ribosomedisplay)是一种体外筛选功能性蛋白质的有力的工具.利用体外转录和翻译偶联系统可以方便而快捷地完成核糖体展示.筛选系统利用一对能够紧密结合的蛋白质:人锚蛋白(ankyrin)和红血球膜带3蛋白细胞质区域(cytoplasmicdomainoferythrocytemembraneproteinBand3,Cdb3)作为模式分子,希望利用cdb3蛋白通过核糖体展示亲和选择得到锚蛋白基因.用于核糖体展示的人锚蛋白基因结构由组装PCR构建,通过PCR技术引入核糖体展示所需的结构元件.在亲和筛选步骤后,只能利用红血球膜带3蛋白筛选得到锚蛋白基因,而不能利用对照牛血清白蛋白(bovineserumalbumin,BSA)筛选得到,从而说明建立的核糖体展示技术能够正常发挥作用.     

人红细胞带3蛋白胞质段的克隆及其表达

人红细胞带3蛋白胞质段(cytoplasmic domain of band 3, cdb3)起着将膜与膜骨架、细胞内环境相联系的重要作用. 以带3蛋白全长基因为模板,用PCR方法扩增出cdb3片段,克隆至pRSET表达质粒上,转化大肠杆菌BL21(DE3). 转化菌经诱导表达出较高含量的cdb3蛋白,纯化后,测得对醛缩酶有抑制活性.     

Mapping the binding site on ankyrin for the voltage-dependent sodium channel from brain.

Erythrocyte ankyrin is a member of a family of proteins that mediate the linkage between membrane proteins and the underlying spectrin-actin-based cytoskeleton. Ankyrin has been shown to interact with a variety of integral membrane proteins such as the anion exchanger, the Na+K(+)-ATPase, and the voltage-dependent sodium channel (NaCh) in brain. To understand how ankyrin interacts with these proteins and maintains its specificity and high affinity for the voltage-dependent NaCh, we have mapped the binding site on ankyrin for the NaCh by examining the binding of purified ankyrin subfragments, prepared by proteolytic cleavage, to the purified rat brain NaCh incorporated into liposomes. 125I-Labeled ankyrin and the radiolabeled 89- and 43-kDa fragments of ankyrin bind to the NaCh with high affinities and with Kd values of 34, 22, and 63 nM, respectively, and have stoichiometries of approximately 1 mol/mol NaCh. The 72-kDa spectrin binding domain is inactive and does not bind to the NaCh. Dissection of ankyrin reveals that the 43-kDa domain retains all the binding properties of native ankyrin to the NaCh. Analysis of the primary structure reveals that the NaCh binding site is confined to a domain of ankyrin consisting entirely of the 11 terminal 33-amino acid repeats and is distinct from the ankyrin domains that interact with spectrin and the Na+K(+)-ATPase.     

Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes

This report presents evidence for diversity in membrane binding sites between three forms of ankyrin: brain ankyrin, erythrocyte ankyrin, and a variant of erythrocyte ankyrin (protein 2.2) present in circulating human erythrocytes that is missing a regulatory domain. These ankyrins were compared with respect to binding to kidney microsomes and exhibited the following behavior. 1) Brain and erythrocyte ankyrin each bind to distinct sites. 2) Protein 2.2 is an activated ankyrin that binds to all of the sites accessible to both brain and erythrocyte ankyrin and, in addition, associates with its own specialized sites. 3) The specificity of these membrane sites for various ankyrins is not absolute but reflects 2.5-10-fold differences in relative affinities. Further evidence that binding sites of different ankyrins share some common features is that the cytoplasmic domain of the erythrocyte anion transporter associates with all three ankyrins and displaces binding of the ankyrin variants to kidney membranes. The differences between erythrocyte and brain ankyrins in association with kidney membranes are likely to have physiological relevance to kidney because immunologically related isoforms of ankyrin are expressed in this tissue: erythroid ankyrin which is restricted to the basolateral domains of two cell types and a brain-related ankyrin expressed in all cells and present on apical as well as basolateral membrane surfaces. An unanticipated observation was the discovery of a membrane-associated ankyrin protease in kidney that is specific for erythrocyte ankyrin and may selectively activate the erythroid isoform of ankyrin. The variety of binding sites within this group of ankyrin proteins supports the idea that ankyrins are capable of linking a number of different membrane proteins to the spectrin-actin skeleton.     

Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3

We have examined the associations of purified red cell band 4.2 with red cell membrane and membrane skeletal proteins using in vitro binding assays. Band 4.2 bound to the purified cytoplasmic domain of band 3 with a Kd between 2 and 8 X 10(-7) M. Binding was saturable and slow, requiring 2-4 h to reach equilibrium. This finding confirms previous work suggesting that the principal membrane-binding site for band 4.2 lies within the 43-kDa cytoplasmic domain of band 3 (Korsgren, C., and Cohen, C. M. (1986) J. Biol. Chem. 261, 5536-5543). Band 4.2 also bound to purified ankyrin in solution with a Kd between 1 and 3.5 X 10(-7) M. As with the cytoplasmic domain of band 3, binding was saturable and required 4-5 h to reach equilibrium. Reconstitution with ankyrin of inside-out vesicles stripped of all peripheral proteins had no effect upon band 4.2 binding to membranes; similarly, reconstitution with band 4.2 had no effect upon ankyrin binding. This shows that ankyrin and band 4.2 bind to distinct loci within the 43-kDa band 3 cytoplasmic domain. Coincubation of ankyrin and band 4.2 in solution partially blocked the binding of both proteins to the membrane. Similarly, coincubation of bands 4.1 and 4.2 in solution partially blocked binding of both to membranes. In all cases, the data suggest the possibility that domains on each of these proteins responsible for low affinity membrane binding are principally affected. The data also provide evidence for an association of band 4.2 with band 4.1. Our results show that band 4.2 can form multiple associations with red cell membrane proteins and may therefore play an as yet unrecognized structural role on the membrane.     

Solution structure of the cytoplasmic domain of erythrocyte membrane band 3 determined by site-directed spin labeling

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.     

Characterization of band 3-ankyrin-Protein 4.2 complex by biochemical and mass spectrometry approaches

The elastic property of red blood cell is supported by interaction between red cell membrane and the intricate cytoskeleton network underlying the membrane bilayer cytoplasmic face. One of the major scaffold protein linkers is band 3-ankyrin complex. Defects occurring in this complex have been found in many inherited diseases, causing red blood cell abnormalities. Here we combined the power of mass spectrometry with conventional biochemical purification methods in order to study the native interactions among band 3, ankyrin and Protein 4.2. This approach provided in vivo evidence for the association between band 3 and N-terminal ankyrin purified directly from the cell membrane. The C-terminal regions of ankyrin were not found to be a stable partner of the band 3 complex. Protein 4.2 was shown here to be an integral part of the complex. Its association to the band 3–ankyrin complex could withstand harsh purification conditions. Our findings lend additional support to the interaction between band 3 and ankyrin N-terminal domain previously shown by in vitro binding assays and provide evidence for a band 3 core complex comprising of band 3, ankyrin and Protein 4.2.     

Molecular epitopes of the ankyrin-spectrin interaction

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

Distance between Cys-201 in erythrocyte band 3 and the bilayer measured by single-photon radioluminescence.

Single-photon radioluminescence (SPR), the excitation of fluorophores by short-range beta-decay electrons, was developed for the measurement of submicroscopic distances. The cytoplasmic domain of band 3 (cdb3) is the primary, multisite anchorage for the erythrocyte skeleton. To begin to define the membrane arrangement of the highly asymmetrical cdb3 structure, the distance from the bilayer of Cys-201 next to the "hinge" of cdb3 was measured by both SPR and resonance energy transfer (RET). cdb3 was labeled at Cys-201 with fluorescein maleimide. For SPR measurements, the bilayer was labeled with [3H]oleic acid. The corrected cdb3-specific SPR signal was 98 +/- 2 cps microCi-1 [mumol band 3]-1. From this and the signal from a parallel sample in which 3H2O was substituted for [3H]oleic acid to create uniform geometry between 3H and the fluorophores, a Cys-201-to-bilayer separation of 39 +/- 7 A was calculated. Confirmatory distances of 40 and 43 A were obtained by RET between fluorescein on Cys-201 and eosin and rhodamine B lipid probes, respectively. This distance indicates that Cys-201 lies near band 3's vertical axis of symmetry and that the subdomain of cdb3 between the hinge and the membrane is not significantly extended. In addition, these results validate SPR as a measure of molecular distances in biological systems.