The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.     

Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel

Brain ankyrin was purified from pig brain membranes in milligram quantities by a procedure involving affinity chromatography on erythrocyte spectrinagarose. Brain ankyrin included two polypeptides of Mr = 210,000 and 220,000 that were nearly identical by peptide mapping and were monomers in solution. Brain ankyrin and erythrocyte ankyrin are closely related proteins with the following properties in common: 1) shared antigenic sites, 2) high-affinity binding to the spectrin beta subunit at the midregion of spectrin tetramers, 3) a binding site for the cytoplasmic domain of the erythrocyte anion channel, 4) a binding site for tubulin, 5) a similar domain structure with a protease-resistant domain of Mr = 72,000 that contains the spectrin-binding activity and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg of membrane protein in demyelinated membranes based on radioimmunoassay with antibody raised against brain ankyrin and affinity purified on brain ankyrin-agarose. Brain spectrin tetramers are present at 30 pmol/mg of membrane protein. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes. Brain ankyrin also may attach microtubules to membranes independently of spectrin and has the potential to interconnect microtubules and spectrin-associated actin filaments.     

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.     

Unexpected complexity in the mechanisms that target assembly of the spectrin cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant beta-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP(2) interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP(2) and the spectrin PH domain codistribute with one another in cells but not with authentic wild type alphabeta-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP(2) binding activity.     

The Molecular Basis for Membrane – Cytoskeleton Association in Human Erythrocytes

Spectrin, the major cytoskeletal protein in erythrocytes, is localized on the inner membrane surface in association with membrane-spanning glycoproteins and with intramembrane particles. The presence of a specific, high-affinity protein binding site for spectrin on the cytoplasmic surface of the membrane has been established by measurement of reassociation of spectrin with spectrin-depleted inside-out vesicles. A 72,000 M_r proteolytic fragment of this attachment protein has been purified, which bound to spectrin in solution and competed for reassociation of spectrin with vesicles. A 215,000 M_r polypeptide has been identified as the precursor of the spectrin-binding fragment. The membrane attachment protein for spectrin was named ankyrin, and has been purified and characterized. Ankyrin has been demonstrated to be tightly associated in detergent extracts of vesicles with band 3, a major membrane-spanning polypeptide, and to bind directly to a proteolytic fragment derived from the cytoplasmic domain of band 3. Ankyrin is thus an example of a protein that directly links a cytoplasmic structural protein to an integral membrane protein. The organization of the erythrocyte membrane has implications for more complex cell types since immunoreactive forms of ankyrin distinct from myosin or filamin have been detected by radioimmunoassay in a variety of cells and tissues. Indirect immunofluorescent staining of cultured cells reveals immunoreactive forms of ankyrin in a cytoplasmic meshwork and in a punctate distribution over nuclei. The staining changes dramatically during mitosis, with concentration of stain at the spindle poles in metaphase and intense staining of the cleavage furrow during cytokinesis.     

Molecular basis and functional consequences of the dominant effects of the mutant band 3 on the structure of normal band 3 in Southeast Asian ovalocytosis

Southeast Asian ovalocytosis (SAO) human red cell membranes contain similar proportions of normal band 3 and a mutant band 3 with a nine amino acid deletion (band 3 SAO). We employed specific chemical modification and proteolytic cleavage to probe the structures of band 3 in normal and SAO membranes. When the membranes were modified specifically at lysine residues with N-hydroxysulfosuccinimide-SS-biotin, band 3 Lys-851 was not modified in normal membranes but quantitatively modified in SAO membranes. Normal and SAO membranes showed different patterns of band 3 proteolytic cleavage. Notably, many sites cleaved in normal membranes were not cleaved in SAO membranes, despite the presence of normal band 3 in these membranes. The mutant band 3 changes the structure of essentially all the normal band 3 present in the SAO membranes, and these changes extend throughout the normal band 3 molecules. The results also imply that band 3 in SAO membranes is present as hetero-tetramers or higher hetero-oligomers. The dominant structural effects of band 3 SAO on the other band 3 allele have important consequences on the functional and hematological properties of human red cells heterozygous for band 3 SAO. Analysis of the altered profile of biotinylation and protease cleavage sites suggests the location of exposed surfaces in the band 3 membrane domain and identifies likely interacting regions within the molecule. Our approach provides a sensitive method for studying structural changes in polytopic membrane proteins.     

The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions

We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.     

Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state

The effects of incubation of erythrocyte ghosts under various conditions (ionic strength or addition of ankyrin, diamines, or ATP) on the lateral motion of band 3 in the membranes were studied by using the fluorescence photobleaching recovery technique. Incubation of ghosts with exogenous ankyrin increased the immobile fraction of band 3, from 0.6 in intact ghosts to 0.8-0.9 when an average of 0.2 mol of extra ankyrin was bound per mole of band 3. Ankyrin-free band 3 proteins were mobile, but their mobility was governed by the spectrin association state in the cytoskeletal network. The diffusion constant was 5.3 X 10(-11) cm2 s-1 at a spectrin tetramer mole fraction of 0.3-0.4 in 10 mM NaCl/5 mM sodium phosphate, pH 7.8, and decreased 1 order of magnitude when the tetramer fraction increased to 0.5 in higher NaCl concentration (150 mM NaCl). A similar decrease was observed when the spectrin tetramer fraction was increased by 0.2 mM spermine in 10 mM NaCl/10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6. On the other hand, the rotational motion of band 3 in the membranes was not affected by the spectrin association state. Trypsin treatment of ghosts cleaved off the cytoplasmic domain of band 3 and caused a marked (8-fold) increase in the lateral mobility, D = 4.0 X 10(-10) cm2 s-1. These results indicate that the lateral mobility of ankyrin-free band 3 protein is restricted by interactions of their cytoplasmic domain with the cytoskeletal network. A model is presented that band 3 can pass the network when spectrins are in dissociated dimers and cannot pass when they are tetramers. The lateral diffusion constant is thus determined by the spectrin dimer population in the network.     

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.     

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.     

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.     

Receptor clustering drives polarized assembly of ankyrin

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.     

Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s)

An assay has been developed to measure association of brain ankyrin with protein site(s) in brain membranes that are independent of spectrin and tubulin, behave as integral membrane proteins, and appear to be similar in several respects to the erythrocyte anion channel. Brain membranes were depleted of ankyrin, spectrin, and other peripheral membrane proteins by a brief incubation in 0.1 M sodium hydroxide. Binding of ankyrin to these membranes fulfilled experimentally testable criteria for a specific protein-protein association. Binding was optimal at physiological values for ionic strength and pH, was of high affinity (Kd = 20-60 nM), and the capacity of 25 pmol/mg of brain membrane protein is in the same range as the number of spectrin tetramers (30 pmol/mg). The membrane-binding site(s) for brain ankyrin are likely to be related in some way to the cytoplasmic domain of the erythrocyte anion channel since binding was inhibited by the anion channel domain and by erythrocyte ankyrin. The binding site(s) for brain ankyrin were released from the membrane by limited proteolysis as active water-soluble fragments capable of inhibiting binding of ankyrin to membranes. Ankyrin-binding fragments of Mr = 40,000 and 68,000 were selectively bound to an erythrocyte ankyrin affinity column. The fragment of Mr = 40,000 is close to the size of the cytoplasmic domain of the erythrocyte anion channel. It is likely based on these results that membrane attachment proteins for ankyrin are present in brain and other tissues and that these membrane proteins have domains homologous at least in conformation to the ankyrin-binding site of the erythrocyte anion channel.     

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.     

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.     

Identification of Contact Sites between Ankyrin and Band 3 in the Human Erythrocyte Membrane

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.     

Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.     

Mapping the ankyrin-binding site of the human erythrocyte anion exchanger

This report describes initial efforts to map the ankyrin-binding site of the cytoplasmic domain of the human erythrocyte anion exchanger. The conclusions are that this site is likely to involve a fairly extended sequence in the midregion of the cytoplasmic domain and requires interactions that are not provided by isolated peptides. The region of the sequence involving residues 174-186 is likely to participate in the ankyrin-binding site based on several experiments. Limited tryptic cleavage in the midregion of the cytoplasmic domain (residues 174 and/or 181) nearly abolished the ability of the cytoplasmic domain to inhibit binding of ankyrin to the anion exchanger. Ankyrin protected the cytoplasmic domain from tryptic digestion. Finally, peptide-specific antibodies against the sequence encompassing the site(s) of tryptic cleavage (residues 174-186) blocked binding of ankyrin to the anion exchanger. However, the sequence comprising the tryptic site is not sufficient for high affinity binding of ankyrin. A 39-amino acid peptide (residues 161-200) that includes the tryptic cleavage site(s) was inactive in inhibiting binding of ankyrin to the anion exchanger. Further evidence for a complex ankyrin-binding site is that peptide-specific antibodies against two different, noncontiguous regions (residues 118-162 and 174-186) both inhibited binding of ankyrin to the anion exchanger and were only 10-20% as effective as antibody against the entire cytoplasmic domain. Finally, the ankyrin-binding site of the anion exchanger did not renature following sodium dodecyl sulfate electrophoresis and transfer to nitrocellulose paper even though spectrin did recover ability to bind ankyrin under the same conditions. Thus, the ankyrin-binding site is not defined by a short continuous sequence. A simple consensus sequence for ankyrin-binding regions in other proteins is not likely.     

Abnormalities of the erythrocyte membrane in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.