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Crystal structure of a 12 ANK repeat stack from human ankyrinR

2002; Springer Nature; Volume: 21; Issue: 23 Linguagem: Inglês

10.1093/emboj/cdf651

1460-2075

Peter Michaely,

Pancreatic function and diabetes

Article1 December 2002free access Crystal structure of a 12 ANK repeat stack from human ankyrinR Peter Michaely Corresponding Author Peter Michaely Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Diana R. Tomchick Diana R. Tomchick Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Mischa Machius Mischa Machius Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Richard G.W. Anderson Richard G.W. Anderson Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Peter Michaely Corresponding Author Peter Michaely Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Diana R. Tomchick Diana R. Tomchick Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Mischa Machius Mischa Machius Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Richard G.W. Anderson Richard G.W. Anderson Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA Search for more papers by this author Author Information Peter Michaely 1, Diana R. Tomchick2, Mischa Machius2 and Richard G.W. Anderson1 1Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75235-9039 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:6387-6396https://doi.org/10.1093/emboj/cdf651 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ankyrins are multifunctional adaptors that link specific proteins to the membrane-associated, spectrin–actin cytoskeleton. The N-terminal, 'membrane-binding' domain of ankyrins contains 24 ANK repeats and mediates most binding activities. Repeats 13–24 are especially active, with known sites of interaction for the Na/K ATPase, Cl/HCO3 anion exchanger, voltage-gated sodium channel, clathrin heavy chain and L1 family cell adhesion molecules. Here we report the crystal structure of a human ankyrinR construct containing ANK repeats 13–24 and a portion of the spectrin-binding domain. The ANK repeats are observed to form a contiguous spiral stack with which the spectrin-binding domain fragment associates as an extended strand. The structural information has been used to construct models of all 24 repeats of the membrane-binding domain as well as the interactions of the repeats with the Cl/HCO3 anion exchanger and clathrin. These models, together with available binding studies, suggest that ion transporters such as the anion exchanger associate in a large central cavity formed by the ANK repeat spiral, while clathrin and cell adhesion molecules associate with specific regions outside this cavity. Introduction Ankyrins are protein adaptors that bridge between the spectrin–actin cytoskeleton and proteins involved in ion transport, cell adhesion and membrane trafficking. Linkages with ion transporters represent the largest class and include associations with the Cl/HCO3 anion exchanger, the Na/Ca exchanger, Na/K ATPase, IP3 receptor, ryanodine receptor and voltage-gated Na channels (Bennett and Stenbuck, 1979; Nelson and Veshnock, 1987; Srinivasan et al., 1988; Joseph and Samanta, 1993; Li et al., 1993; Bourguignon et al., 1995; Festy et al., 2001). These linkages serve to anchor the spectrin–actin cytoskeleton on the membrane and to concentrate these transporters in specialized membrane domains (reviewed in Bennett and Baines, 2001). The ankyrin linkages with cell adhesion molecules such as the L1 family (Davis et al., 1993) enhance cell adhesion and facilitate the incorporation of cells into tissues (Hortsch et al., 1998; More et al., 2001). Ankyrins also bind clathrin and participate in membrane trafficking at the level of both coated pit budding (Michaely et al., 1999) and trafficking of specific membrane proteins (Tuvia et al., 1999). Metazoan cells employ a large number of ankyrin isoforms for these different functions. Vertebrates have three ankyrin genes (ank1–3) that code for three families of proteins (ankyrinR, B and G, respectively). Most ankyrin isoforms contain two conserved domains that mediate protein–protein interactions. A central, 'spectrin-binding' domain associates with β-spectrin, while the N-terminal, 'membrane-binding' domain binds most other proteins. Isoform specificity is determined by the variably spliced C-terminal domain, which contains isoform- specific targeting information and regulatory functions that modulate the binding activities of the two protein interaction domains (reviewed in Bennett and Baines, 2001). Of fundamental importance to ankyrin function is the extraordinary ability of the membrane-binding domain to bind many unrelated proteins. This region is highly conserved and consists of 24 copies of the ANK repeat motif (also referred to as ankyrin repeats). ANK repeats are a common motif whose L-shaped structure consists of two α-helices and a long loop. Typically, repeats stack in a linear fashion to form curved assemblies that contain between four and seven repeats and associate with a single protein target (reviewed in Sedgwick and Smerdon, 1999). Ankyrins, in contrast, contain 24 repeats that interact with at least 10 distinct proteins. Proteolytic mapping suggests that the 24 repeats are divided into four smaller domains (D1, D2, D3 and D4) of six repeats each; however, it is not clear how these subdivisions relate to protein associations since many binding sites overlap the domain boundaries (Michaely and Bennett, 1993, 1995b). Herein, we have determined the crystal structure of the D34 region of human ankyrinR, which consists of repeats 13–24 and a small portion of the spectrin-binding domain. We find that the 12 ANK repeats in D34 are stacked in the form of a contiguous, left-handed superhelix. The spectrin-binding domain segment is extended and interacts with the repeat stack in a peptide-in-groove association. The structural information has been used to model the 24 repeats of the entire membrane-binding domain and the associations of the D34 region with the clathrin heavy chain and the Cl/HCO3 anion exchanger. We propose that the membrane-binding domain forms a large, spiral, hook-like structure. Ion transport proteins such as the anion exchanger may associate with the concave surface of this hook, while clathrin and cell adhesion molecules decorate the outside of the helical hook. Results Several regions of the membrane-binding domain of human ankyrinR were utilized in crystallization attempts. As has been seen in other ANK repeat proteins (Michel et al., 2001), both the membrane-binding domain and the proteolytically defined subdomains were prone to aggregation. Inclusion of residues 798–827 from the spectrin-binding domain markedly improved protein solubility. Crystallization efforts therefore concentrated on constructs bearing residues 1–827 (D1234), 402–827 (D34) and 600–827 (D4) (Figure 1A). Small, poorly diffracting crystals were obtained initially with the D34 construct. Re-engineering of the D34 construct to include six glycine residues at the N-terminus improved protein solubility and crystal quality. Optimization of the crystallization conditions generated large crystals that exhibited the symmetry of space group P6322 with one molecule per asymmetric unit. The crystal structure was solved by the multiwavelength anomalous dispersion (MAD) method using a seleno-methionine (SeMet) variant of D34 (Table I). The final refined model of the native protein contains residues 405–797 and 802–812 of human ankyrinR (Table I). Figure 1.Structure of the D34 region of ankyrinR. (A) Ankyrins typically are composed of four domains: an ANK repeat bearing, 'membrane-binding' domain, a central 'spectrin-binding' domain, a death domain (DD) and a C-terminal regulatory domain. The D34 region of ankyrinR contains ANK repeats 13–24 and a short portion of the spectrin-binding domain. Cylinders indicate the positions of the two helical segments in each repeat. Amino acids are given as one-letter code, where Ψ indicates a non-polar residue. Position numbering within repeats is based upon the exon–intron boundaries of the ank1 gene. The amino acid sequence of the D34 region is shown, with the repeat designation on the left and the amino acid number on the right of the sequence block. (B) Stereo view of D34. Individual repeats are rainbow colored such that repeat 13 is red and repeat 24 is violet. (C) Surface labels are given for the repeat stack. The bottom image is rotated 90° relative to the top image. Download figure Download PowerPoint Table 1. Data collection, structure determination and refinement Data collection Crystal Native SeMet SeMet Energy (eV) 12 661.1 12 659 (peak) 12 658.7 (inflection) Resolution range (Å) 50.0–2.50 99.0–2.79 99.0–2.79 Total observations 415 031 409 137 220 227 Unique reflections 37 994 25 576 24 337 Data completeness (%) 98.4 (93) 89.9 (44.2) 85.2 (35) Rmerge (%)a 8.2 (69.9) 9.1 (57.9) 8.4 (70) I/σ(I) 22.6 (1.3) 29.4 (1.7) 21.3 (1.6) Anomalous scatterer 8 Se sites Figure of merit (acentric/centric/overall) 0.256/0.171/0.243 Resolution range (Å) 30.0–2.70 Wilson B-factor(Å2) 67 No. of reflections Rwork/Rfree 29 145/1155 Atoms (non-H protein/halide) 3085/10 Rwork (%) 31.9 Rfree (%)b 30.3 R.m.s.d. bond length (Å) 0.01 R.m.s.d. bond angle (°) 1.62 Mean B-value (Å2) (main chain/side chain/halide) 113.8/114.4/129.3 Cross-validated σA-coordinate error (Å) 1.07 Missing residues 4 (798–801) No. of alternative conformations 3 Data for the outermost shell are given in parentheses. a Rmerge = , where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent observations of each unique reflection. b Rfree is calculated for a randomly selected 3.7%. Structure of D34 The structure of D34 (Figure 1B and C) consists of a contiguous stack of 12 ANK repeats (repeats 13–24 of the membrane-binding domain) plus a short extended segment from the start of the spectrin-binding domain. Individual ANK repeats have the typical 'L'-shaped structure seen in ANK repeats from non-ankyrin proteins (Sedgwick and Smerdon, 1999). The 'L' structure consists of two short, antiparallel α-helices (positions 4–11 and 15–23 of the ANK repeat consensus sequence) followed by a long loop (positions 24–30). Individual repeats are linked by a β-hairpin that contains amino acids at positions 31–33 from one repeat and positions 1–3 of the next repeat (Figure 2A). The repeats generate a linear stack such that the α-helices of adjacent repeats form a four-helical bundle (Figure 2B). The long loop covers one end of the helical bundle, while the β-hairpin extends away from the bundle, perpendicular to the axis of the first helix and the repeat stack. At the C-terminus of the repeat stack, the spectrin-binding domain segment adopts an extended conformation that folds back and binds in a cleft formed by repeats 20–24. Figure 2.Consensus residues of the ANK repeat. (A) The conformation of conserved residues of the ANK repeat consensus sequence is shown, oriented along the axis of the repeat stack. The α-helical segments are depicted as ribbons (green), and the non-helical segments are depicted as a C- trace (light gray). Conserved histidines are blue, aspartates and asparagines are red, threonines are yellow, and aliphatic residues are black. Each repeat has four solvent-exposed surfaces as indicated. (B) The helical bundle core between repeats 16 and 17 is shown. Shown in light gray are Ala510, Ala511, Val519 and Val551 at positions 9, 10 and 18 of repeat 16 and position 17 of repeat 17, respectively. These residues are situated below the dark gray shaded Leu507, Leu523 and Leu555 at positions 6 and 22 of repeat 16 and position 21 of repeat 17, respectively. An image of the D34 structure is given between the panels for reference. Download figure Download PowerPoint The repeat stack All repeats within D34 have 33 amino acids and show strong homology with the consensus sequence, -G-TPLHΨAA--GH--ΨV-ΨLL--GA--D/N---, where Ψ designates aliphatic residues. Table II and Figure 2B summarize how the consensus residues define the boundaries of the two helices, stabilize the conformation of individual repeats and form the interfaces between repeats. Table 2. Selected non-core contacts Intra-repeat interactions Position Common residue Atoms ↔ Position Common residue Atoms Contact type 4 Thr sc 7 His sc H-bond 4 Thr sc 7 His N H-bond 7 His sc 30 Non-polar O H-bond 27 Polar sc 28/29 Varies mc H-bond Repeat n Repeat n + 1 Position Common residue Atoms ↔ Position Common residue Atoms Contact type 7 His sc 3 Varies O H-bond 11 Varies mc 8 Non-polar sc vdW 10/11 Varies O 14 His sc H-bond 18/22 Non-polar mc 20 Non-polar sc vdW 28 Non-polar sc 26 Ala sc vdW 29 Asx sc 27 Polar N H-bond 32 Varies O 2 Varies N H-bond 32 Varies N 3 Varies O H-bond 33 Varies N 1 Varies O H-bond Contacts of the hydrophobic cores of helical bundles have been excluded; see Figure 2B. vdW, van der Waals contact; H-bond, hydrogen bond contact; sc, side chain; mc, main chain; O, carbonyl oxygen; N, amide nitrogen. The threonine at position 4 of the consensus sequence (Thr[4]), Pro[5], Gly[13], Gly[25] and Ala[26] determine the boundaries of the helices. Thr[4] is the N-terminal capping residue of the first helix and frequently forms a hydrogen bond with the backbone amide of the third residue of the helix. Pro[5] starts the first helix most probably because its dihedral angle (φ) is restricted by the proline ring to a value near the optimal φ for helices (Aurora and Rose, 1998; Richardson and Richardson, 1988). Gly[25] and Ala[26] constitute a common C-terminal helix cap (Edwards et al., 1987). Glycines generally are considered to be helix-disrupting residues and Gly[13] most probably promotes the formation of the short loop that connects the first and second helices. Residues at positions 9, 16, 24, 27 and 30 appear to stabilize the fold of individual repeats. Positions 16 and 24 frequently contain negative and positive charges, respectively, and probably serve to stabilize the second helix by neutralizing its dipole moment (Shoemaker et al., 1987). Ala[9] and hydrophobic residues at position 30 appear to help orient the two helices relative to each other. Position 27 stabilizes the unusual conformation of the long loop that separates the second helix from the β-hairpin by forming a hydrogen bond to the backbone amide of position 29. Residues at many conserved positions form the inter-repeat contacts that join adjacent repeats to the repeat stack. The contacts made by these positions can be classified into three groups. Centermost are the van der Waals contacts made by residues that form the hydrophobic core of the four-helix bundles. These residues include Leu[6], Ala[10], Leu[21], Leu[22] and mid-sized hydrophobic residues at positions 17 and 18 (Figure 2B). A second layer of van der Waals contacts made by hydrophobic residues at positions 8, 20, 26 and 28 surrounds this central core (Table II). Finally, an outer layer of hydrogen bonds formed by His[7], His[14], Asn/Asp[29] and residues of the β-hairpin provide additional inter-repeat connections (Table II). The 12 ANK repeats in D34 stack side by side to form a 125 Å long, left-handed superhelix (Figure 1B). Each repeat is twisted counter-clockwise 2–3° relative to the preceding repeat and contributes 13° of pitch to the superhelix. Equivalent residues in each repeat of the super helix form four distinct surfaces on the repeat stack. These surfaces consist of (i) a concave 'bottom' surface using residues at positions 13–16; (ii) a concave 'ankyrin groove' surface (Sedgwick and Smerdon, 1999) using positions 1, 3, 11, 12 and 32; (iii) a convex 'back' surface comprising positions 19, 23 and 24; and (iv) a broad, convex 'top' surface consisting of positions 27, 29, 31 and 33 (Figures 1C and 2A). The ankyrin groove surface is directed towards the axis of the superhelix, while the junction between the top and back surfaces is directed away from this axis. Residues that form these surfaces are solvent exposed and are in variable positions of the consensus sequence. The first and last repeats have additional 'end' surfaces that contain hydrophilic residues at consensus positions that are typically hydrophobic and protected from solvent in the helical bundles of the inner repeats. Residues in the helical bundle cores control the superhelical curvature of the repeat stack. The ankyrin groove exhibits concave curvature because the helices of the inner row, consisting of the first helices in successive repeats, pack more tightly than the helices in the second, outer row. The packing difference is due largely to residues at positions 10, 17 and 18. The small side chains of residues at position 10 allow the first helices to pack closer than the mid-sized residues at positions 17 and 18 in the second helices (Figures 1C and 2). The curvature about the bottom surface is due to equivalent positions in successive repeats being farther apart near the top surface than at the bottom surface. The shorter spacing at the bottom is determined by the relatively small side chains of residues at positions 10, 17 and 18 as compared with the larger leucines at positions 6, 21 and 22 at the top (Figures 1C and 2). ANK repeat interactions In the D34 structure, the ankyrin groove surface of repeats 20–24 associates with the first 11 residues of the spectrin-binding domain. This 11 residue segment binds in an extended conformation, antiparallel to the repeat stack (Figures 1B and 3). The interaction is mediated by a number of van der Waals contacts involving His802, Met804, Pro807 and Val810, as well as polar interactions involving Ser805, Glu808 and Asp811. Ser805 plays a central role, forming both main chain and side chain hydrogen bonds with Tyr702, Gln740 and Gln743 on the repeat stack. Figure 3.Spectrin-binding domain fragment. Shown is a stereo pair of the interaction between residues 802–812 and the repeat stack. Residues 802–812 are shown as sticks on the ankyrin groove surface of repeats 19–24. Atoms are colored gray for carbon, red for oxygen, blue for nitrogen and green for sulfur. The displayed surface is colored by electrostatic potential from −10 kT (red) to +10 kT (blue). The electrostatic surface potential was calculated and rendered in the program GRASP (Nicholls et al., 1991). Download figure Download PowerPoint The D34 region of ankyrins associates with at least five proteins: the clathrin heavy chain, the Cl/HCO3 anion exchanger, Na/K ATPase, the L1 family of cell adhesion molecules and sodium channels of the voltage-gated and amorilide-sensitive family (Srinivasan et al., 1992; Michaely and Bennett, 1995a, 1995b; Thevananther et al., 1998; Michaely et al., 1999). These proteins do not share sequence homology and, therefore, do not use a common motif for D34 binding. In the following, we have focused on the associations with clathrin and the Cl/HCO3 anion exchanger (band 3), because structural information exists for the ankyrin-binding regions of these two proteins (ter Haar et al., 1998; Zhang et al., 2000). The association of D34 with the cytoplasmic domain of the anion exchanger (CDB3) is well characterized. Antibody competition and protein chimera studies indicate that the interaction surface on CDB3 involves residues 1–75, 118–141, 155–160 and 174–193 (Davis et al., 1989; Willardson et al., 1989). These residues form a slightly convex surface on the CDB3 dimer, which folds as a seven-stranded α/β sandwich and dimerizes by means of a shared α-helical motif (Zhang et al., 2000). Both repeats 13–18 and 22–23 of D34 are required for binding to CDB3 (Davis et al., 1991; Michaely and Bennett, 1995a). The ankyrin interaction with clathrin is less well characterized. Previously, we identified an interaction between repeats 19–24 of ankyrin (D4) and the N-terminal domain of clathrin heavy chain (CTD) (Michaely et al., 1999). The CTD contains a β-propeller-like arrangement of seven β-sheets followed by an extended series of α-helices. To map the ankyrin-binding site better on the CTD, we expressed various truncated forms of the CTD and tested their ability to interact with D4 using a far-western assay system (Davis et al., 1991; Michaely and Bennett, 1993) (Figure 4A). The entire β-propeller region (residues 1–332) of the CTD was required to interact with D4. Removing as few as 14 amino acids from the N-terminus was sufficient to abolish binding. Deletion of residues from either terminus disrupts the final β-sheet and may also affect the normal organization of the β-propeller. Figure 4.Binding site mapping. (A) The upper portion presents far-western blots (see Materials and methods) showing the relative ability of GST fusions with clathrin fragments to associate with the D4 region. The numbers above each lane indicate the amino acids of clathrin present in each fusion. The lower portion of the panel provides a pictorial representation of these regions. On the left is an amino acid marker together with a diagram indicating the location of the β-propeller (box) and α-helical (line) segments of the CTD. (B) In vitro binding assays were performed using 50 nM 125I-labeled CTD1–494, D34- (closed circles) or ARH- (open circles) coupled Sepharose beads, and increasing concentrations of CDB3 (closed circles) or D34 (open circles). ARH (autosomal recessive hypercholesterolemia protein) contains a canonical clathrin box sequence, while the D34 region does not. All assays were carried out in triplicate with standard error shown with error bars. Values shown represent specific binding only. Download figure Download PowerPoint Clathrin also binds proteins that contain the clathrin box sequence, LL(D/N)L(D/E), via a cleft on the β-propeller between the first and second β-sheets. To determine whether D34 binds near this region, we tested the ability of D34 to inhibit CTD binding to the clathrin box containing autosomal recessive hypercholesterolemia protein (ARH). Figure 4B shows that D34 is a good competitive inhibitor of CTD binding to ARH. In contrast, CDB3 did not compete with CTD for D34 binding. These results suggest that the D34-binding site on clathrin is near the first and last β-sheet of the β-propeller, while the clathrin-binding site on D34 involves repeats 19–24. We used a protein-docking program suite, 3D-Dock, to model the interactions of the D34 region with CDB3 and the β-propeller of clathrin (Gabb et al., 1997). Potential docking modes were identified based upon surface and charge complementarity. Probable models were then checked against steric and biochemical criteria. The model with the highest score for the clathrin association is shown in Figure 5A. The proposed contact is between the first and last β-sheets of the CTD and the top surface of the last three repeats of D34. The interface shows a high degree of charge complementarity consistent with the observation that the interaction is sensitive to salt. We used a similar approach to look at potential interactions between D34 and CDB3. The modeling suggests that bottom and ankyrin groove surfaces of the ANK repeat stack associate with the CDB3 dimer on the surface opposite the dimer interface (Figure 5B). The proposed contact surface on CDB3 consists of the third helix and the loops that connect the first and second β-strands, the fifth β-strand and the second helix, the sixth and seventh β-strands, the sixth helix and the eighth β-strand, and the ninth and tenth β-strands. This interaction surface buries >1500 Å2 and contains few potential salt bridges, which is in agreement with a high affinity interaction between D34 and CDB3 that is not salt sensitive (Michaely and Bennett, 1995a). Figure 5.Modeled binding partners. (A) The model for the complex of repeats 19–24 of D34 (green) with the CTD (purple). (B) The association of D34 (green) with the CDB3 dimer (gray and purple). Residues predicted to bind ankyrin are shown on CDB3 in purple. Download figure Download PowerPoint Modeling of the ankyrinR membrane-binding domain In addition to interactions made by repeats 13–24 of the D34 region, several associations are known to require repeats 1–12. We have modeled the entire set of 24 repeats by extending the D34 superhelix to include all 24 ANK repeats, retaining the 13° pitch for each repeat. The model predicts that the entire membrane-binding domain forms a superhelical spiral with an axial length of 132 Å and radius of 45 Å (Figure 6A). The 800 residues of the full repeat stack provide a surface area of >27 000 Å2 for protein interactions. The wide spiral of the modeled membrane-binding domain has a more globular structure than the D34 construct, consistent with previously published hydrodynamic measurements (Michaely and Bennett, 1993). One interaction made by repeats 7–12 (D2) is an association with a second CDB3 dimer (Michaely and Bennett, 1995a). In Figure 6B, we extended the D34–CDB3 model to encompass all 24 repeats of the membrane-binding domain and both dimers of the CDB3 tetramer. Significantly, the combined model positions the D2 region so that it can associate with the second CDB3 dimer in the tetramer. The D2 association is salt sensitive, and the modeled association between D2 and the second CDB3 dimer includes several potential salt bridges. Figure 6.Membrane-binding domain. (A) A stereo view of a model of the membrane-binding domain viewed along the spiral axis with the C-terminus closest to the viewer. Repeats are rainbow colored such that repeat 1 is red and repeat 24 is violet. (B) An extension of the D34–CDB3 model to include all 24 repeats of the membrane-binding domain and all four subunits of the CDB3 tetramer. The membrane-binding domain is shown as a green ribbon, while the four subunits of the tetramer are shown as red, blue, cyan and pink ribbons. The D34 region of the membrane-binding domain interacts with the red subunit. Download figure Download PowerPoint Discussion We have determined the crystal structure of ANK repeats 13–24 and a small portion of the spectrin-binding domain of human ankyrinR. The 11 amino acid portion of the spectrin-binding domain binds through a peptide-in-groove interaction with the C-terminal ANK repeats. ANK repeats provide the principle sites of protein interaction on ankyrins, and we modeled the interactions of D34 with the clathrin heavy chain and CDB3. In D34, the individual ANK repeats adopt the typical 'L'-shaped conformation similar to that observed in structures of ANK repeats found in non-ankyrin proteins (reviewed in Sedgwick and Smerdon, 1999). Models based upon biochemical and hydrodynamic data had suggested that the 12 ANK repeats of the D34 region of ankyrinR were divided into two subdomains of six repeats each. The D34 structure now reveals that these 12 ANK repeats form a contiguous extended stack that provides an extensive surface area for protein interactions. A striking feature of the ANK repeats in ankyrinR is their uniformity in both amino acid sequence and three-dimensional structure. Most repeats in ankyrins and all repeats in the D34 region consist of exactly 33 amino acids with strong adherence to the consensus sequence. Because most consensus residues provide inter-repeat contacts, the strong conservation with the repeat consensus sequence results in extensive and uniform connectivity between repeats (Figures 1 and 2; Table II). Extensive connectivity may be required for ankyrins to fulfill their role as the primary linker protein between the spectrin–actin cytoskeleton and membranes. Mechanical deformations of cells and tissues most probably place stress on this linkage. The abundance of inter-repeat contacts may protect the repeat stack from mechanical unfolding. In addition, the uniform nature of the inter-repeat contacts may allow differential splicing to remove individual repeats without compromising the integrity of the repeat stack. All ANK repeat-encoding exons of vertebrate ankyrins have either one or two repeats with exon boundaries exactly in register with position one of the repeat consensus sequence (Gallagher et al., 1997). Because ANK repeats form protein-binding sites using multiple repeats, deletion of individual repeats by alternative splicing has the potential to produce novel binding sites analogous to V(D)J joining in immunoglobulin genes (reviewed in Grawunder et al., 1998). A second distinctive feature of the ANK repeats of D34 is the curvature of the repeat stack. ANK repeat stacks of different proteins have unique curvatures that presumably are adapted to associate best with specific protein ligands. Comparison of r.m.s. deviations between individual repeats of several proteins shows that curvature is