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Structure of the Armadillo Repeat Domain of Plakophilin 1
doi:10.1016/j.jmb.2004.11.048
J. Mol. Biol. (2005) 346, 367–376
Structure of the Armadillo Repeat Domain of Plakophilin 1 Hee-Jung Choi and William I. Weis* Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine 299 Campus Drive West Stanford, CA 94305-5126 USA
The p120ctn subfamily of armadillo domain proteins has roles in modulating intercellular adhesion by cadherin-containing junctions. We have determined the crystal structure of the arm repeat domain from plakophilin-1 (PKP1), a member of the p120ctn subfamily that is found in desmosomes. The structure reveals that the domain has nine instead of the expected ten arm repeats. A sequence predicted to be an arm repeat is instead a large insert which serves as a wedge that produces a significant bend in the overall domain structure. Structure-based sequence alignments indicate that the nine repeats and large insert are common to this subfamily of armadillo proteins. A prominent basic patch on the surface of the protein may serve as a binding site for partners of these proteins. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: adherens junctions; desmosomes; armadillo proteins; plakophilins; p120ctn
Introduction Armadillo (arm) repeat proteins mediate protein– protein interactions in diverse cellular processes, including cell junction assembly, nuclear transport, and transcriptional activation. Proteins in this family, which includes the adherens junction component and transcription factor b-catenin, and the nuclear import protein importin-a, contain multiple copies (typically 7–13) of a 42 amino acid residue repeat motif.1 Each repeat consists of three a helices, designated H1, H2, and H3.2 Successive arm repeats pack together to form an elongated superhelical structure. Structural studies of b-catenin3–11 and importin-a12–17 have shown that many of their ligands bind in the groove formed by the superhelical structure of the arm repeat domain. Several different arm proteins are present in cadherin-containing intercellular junctions (Figure 1). In adherens junctions, b-catenin binds to the cytoplasmic domain of classical cadherins. This complex is in turn linked to the actin cytoskeleton by a-catenin. Plakoglobin, which is highly homologous to b-catenin, is found in both adherens junctions and desmosomes. Plakoglobin can replace b-catenin in adherens junctions, where it binds to cadherins and to a-catenin. In desmosomes, plakoglobin interacts with the desmosomal Abbreviations used: Arm, armadillo; PKP, plakophilin. E-mail address of the corresponding author: [email protected]
cadherins, desmogleins and desmocollins. The plakoglobin/desmosomal cadherin complex is connected to intermediate filaments through desmoplakin. The protein p120ctn and several other homologous proteins form a subgroup of junctional arm proteins whose sequences are distinct from those of b-catenin and plakoglobin.18 These proteins contain a central arm repeat domain predicted to have ten arm repeats, flanked by N and C-terminal regions of varying sizes. p120ctn associates with cadherin/ catenin complexes19–21 and appears to modulate adhesion.18,22 The arm repeat region of p120ctn binds to a juxtamembrane site in the cytoplasmic domain of cadherins distinct from that of b-catenin,23–25 thereby forming a ternary complex with b-catenin and E-cadherin. The precise role of p120ctn is unclear, but it may influence the ability of cadherins to form cell-surface dimers thought to be the functionally adhesive unit.24–26 Recent evidence indicates that p120ctn has a role in the trafficking of cadherins to developing adherens junctions and in cadherin turnover.27–30 p120ctn may serve in regulating cytoskeletal assembly at junctions by virtue of its interactions with Rho-family GTPases.31–33 Plakophilins (PKPs) are p120ctn relatives found in desmosomes. Several types of PKPs have been detected at different cellular locations or at different stages of development. PKP1 is expressed predominantly in the suprabasal layers of stratified and complex epithelia, 34 whereas PKP2 is expressed in all simple, complex and stratified
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
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Plakophilin 1 Armadillo Repeat Domain Structure
Figure 1. A diagram of adherens junctions and desmosomes. Armadillo repeat proteins are shown in colors, with blue representing b-catenin and plakoglobin, and orange/red representing p120ctn and plakophilin. In adherens junctions, p120ctn binds to the cytoplasmic domain of classical cadherins. Plakophilins are desmosomal proteins thought to participate in the linkage between the desmosomal cadherin–plakoglobin complex and desmoplakin.
epithelia as well as non-epithelial tissues such as myocardium and lymph node follicles.35 PKP3 is found at simple and almost all stratified epithelia except for hepatocytes and hepatocellular carcinoma cells and it is expressed uniformly in epidermal layers.36,37 PKP4, which is known also as p0071, is expressed in endothelial cells and it is unique amongst the p120ctn subfamily, in that it is found in both adherens junctions and desmosomes.38 All PKPs have been found in the nucleus as well as at cell junctions, but the significance of this observation remains unclear. The arm repeat domains of b-catenin, plakoglobin, and p120ctn mediate the interactions of these proteins with many of their binding partners. In contrast, the N-terminal head domain of PKPs appears to be responsible for the interaction with their desmosomal partners, including desmoplakin, desmocollins, desmogleins, and intermediate filaments.39–42 Indeed, the arm domain of p0071 is required for adherens junction localization, whereas its N-terminal head region is responsible for desmosomal targeting.43 The arm domain of PKP1 has been observed to associate with actin filaments, suggesting that PKP1 may be involved in the regulation of the actin cytoskeleton.41 However, yeast two-hybrid analysis showed that the arm domain of PKP1 did not interact with actin monomer,41 and cosedimentation assays using purified F-actin filaments showed that PKP1 did not associate with actin filaments in vitro (our unpublished results), implying that other PKP1 interacts with the actin cytoskeleton through other actin-binding proteins. As more information emerges about the modulatory roles of p120ctn and its relatives in cell adhesion, structural data for this distinct subgroup of arm proteins will be important for understanding the molecular basis of their interactions. Here, we present the first arm domain structure from the p120ctn family, that of PKP1. The structure shows significant differences with previously solved arm
domain structures, and demonstrates that this subfamily contains nine rather than the predicted ten arm repeats. A prominent basic groove formed by the structure likely represents a ligand-binding surface.
Results and Discussion Overall structure Structure-based sequence alignments of PKP1 indicate that residues 237–704 correspond to the arm repeat domain. This region was expressed in Escherichia coli, purified, and crystallized. The ˚ resolution by singlestructure was solved at 2.8 A wavelength anomalous dispersion phasing of selenomethionyl protein. The final model comprises amino acid residues 243–387, 396–480, and 509–700. The PKP1 arm region structure reveals a single domain with nine repeats (Figure 2). In contrast to other known arm repeat structures, the PKP1 domain is sickle-shaped rather than straight, with ˚ . The diameter of a crossan overall length of 95 A ˚ . A long, section through the repeats is about 32 A flexible insert containing residues 464–524 is present between the fifth and sixth arm repeats. This region does not disrupt the continuous packing of the repeats, but is the site of a major bend in the arm domain. The individual PKP1 arm repeats are very similar to those found in other arm domain structures. Each repeat is composed of three helices, H1, H2 and H3, with linkers of highly variable length between H2 and H3. Highly conserved residues mediate the packing interactions between neighboring repeats and within single repeats (Figure 3). Most conserved hydrophobic residues in H3 are involved in intra-repeat packing or hydrophobic contacts with the next repeat. In particular, H3 has a very highly conserved LxNL sequence motif near its C
Plakophilin 1 Armadillo Repeat Domain Structure
Figure 2. The overall structure of the PKP1 arm repeat domain.
terminus. The two leucine residues participate in intra-repeat hydrophobic interactions with conserved hydrophobic residues in the middle of H2 and H1, respectively (Figure 3). Other hydrophobic residues conserved in H2 and H3 mediate interrepeat packing interactions to form a contiguous hydrophobic core throughout the domain. Among
369 three conserved Leu residues in H2, the first and the third interact with the hydrophobic residues of the next repeat, and the second, which is located at the opposite side of helix H2, interacts with the previous repeat. All repeats except R6 have similar secondary structural elements. R6, which is followed by the insert, does not have an H1 (Figures 2–4). Instead, the space that would be occupied by H1 in a standard arm repeat is taken by a part of the repeat 5–6 insert, which forms contacts distinct from those normally made by H1 (see below). Nonetheless, H2 and H3 of R6 align well with the corresponding regions of other repeats. Even though the arm repeats of PKP1 are similar, there are some variations in the position of H1 relative to H2 and H3. The angles between H1 and H2 vary from 558 to 1058, as compared with the smaller variation of 358 to 558 found in the H2–H3 angles (Table 1). The variability in the position of H1 relative to H2 and H3 correlates with sequence variations. Repeats 2, 3, 5 and 7 have at least one glycine residue between H1 and H2, which makes a sharp bend between H1 and H2 of about 708 to 908. In the repeats lacking this glycine residue, the angles between H1 and H2 are more variable, ranging from 578 to 1008. Because H1 is connected to H3 of the previous repeat by a very short turn composed of one to three amino acid residues, the position of H1 affects the relative location of the
Figure 3. The structure-based sequence alignment of PKP1 and p120ctn arm domains. PKP1 residues comprising H1, H2, and H3 in each repeat are boxed; the green box indicates the 310 helix in the insert. Italicized PKP1 residues are disordered in the structure. Conserved residues that define the arm consensus motif are highlighted in yellow, and the consensus is shown at the bottom of the figure. In the consensus, the single-letter code is listed if the residue is present in at least two-thirds of the repeats. Consensus positions with large or small hydrophobic residues are shown as filled or open boxes, respectively, and those with either small or large hydrophobic residues as half-filled boxes. The C sign indicates a positively charged residue. For comparison, the consensus arm repeat motif derived from the b-catenin structure2 is indicated at the bottom. Conserved residues that mediate contacts between the insert and repeats 5–7 are highlighted in blue.
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Plakophilin 1 Armadillo Repeat Domain Structure
An insert replaces a predicted arm repeat
Figure 4. Interactions of the repeat 5–6 insert. Repeats 5–7 are colored as in Figure 2, and H1, H2 and H3 in each repeat are indicated. The insert is shown with a grey backbone, except for the 310 helix containing Leu522, which is indicated by the dark grey backbone. Key residues that mediate the interactions are shown in balland-stick representation. Carbon, nitrogen, oxygen, and sulfur atoms are shown as yellow, blue, red, and green spheres, respectively. Salt bridges between Glu468 and Arg420 and Arg424 are shown as broken black lines.
previous repeat. This can be seen in the rotation and translation between neighboring repeats of PKP1, ˚ to 9.5 A ˚, which vary from 108 to 308 and from 8.5 A respectively. Despite the variation in the relative positions of H1 amongst the repeats, the packing interactions mediated by the conserved hydrophobic residues of H1 are preserved throughout the structure.
Table 1. Inter-helix angles within PKP-1 arm repeats Helix angle (8) Repeat R1 R2 R3 R4 R5 R6 R7 R8 R9 AverageGSD
H1–H2
H2–H3
N/A 78.5 67.9 55.6 79.5 N/A 93.6 58.3 104.7 76.9G18.0
40.8 35.6 32.7 47.1 46.1 35.4 40.1 37.6 54.5 41.1G7.0
On the basis of the sequence alignments of b-catenin and p120ctn-family proteins,18 part of the 61 amino acid residue insert between repeats 5 and 6 was expected to form an arm repeat. Instead, this region starts as two a-helices that protrude outward from the main body of the structure, and are interrupted by a conserved proline residue. These helices are followed by a flexible loop of 28 residues (italics in Figure 3), and the insert ends as a 310 helix of seven residues. The central portion of the insert is not visible in the electron density map and therefore unstructured, but the beginning and ends of the insert are structured and stabilized by interactions with conserved residues in repeats 5 and 7 (Figure 4). Glu468, which is located at the beginning of the insert and is highly conserved in all p120ctn family members, forms salt bridges with the conserved, positively charged residues Arg420 and Arg424 in R5 (Figure 4). Leu465, Val469, and Tyr473 at the beginning of the insert, and Leu522 and Tyr523 at the end, pack with Met561 and Met565 of R7 (Figures 3 and 4). Although most of the insert sequence is not conserved in p120ctn family members, the residues of the loop that interact with the main body of the protein are highly conserved, implying that other p120ctn homologs have a similar structure in this region. Therefore, we predict that all members of the p120ctn subfamily of arm proteins will have nine instead of ten arm repeats, and the numbering of the repeats used here reflects this fact. Evidence for an insert rather than a repeat at residues 589 to 661 of p120ctn comes also from the observation that deletion of this sequence does not affect binding to E-cadherin, whereas the flanking arm repeats are required for this interaction.44 Comparisons with other structures containing a-helical repeats The structures of two other arm repeat domain structures, b-catenin 2 and importin-a 12,13 are known, as are several other a-solenoid structures composed of helical repeats such as HEAT45–47 and Pumilio motifs.48,49 None of these structures has a repeat-length insert between neighboring repeats such as that present in the PKP1 arm domain. It is interesting to note that the large bend in PKP1 that arises from this insert prevented determination of the structure using molecular replacement phasing from known arm domain models. Even though the overall structures of arm domain and HEAT repeat-containing proteins are somewhat different, it has been noted that they likely have a common origin.1 The main feature that distinguishes these types of repeat is that arm repeats contain three helices, H1, H2 and H3, whereas HEAT repeats have just two helices, denoted A and B.45,46 Helix A of the HEAT repeat is usually bent, and corresponds to H1 and H2 of an arm repeat, and helix B corresponds to arm repeat
Plakophilin 1 Armadillo Repeat Domain Structure
H3. The arm and HEAT repeats share the conserved hydrophobic residues that mediate inter-repeat and intra-repeat interactions. Structural alignment between H2 and H3 of PKP1 and the corresponding regions of b-catenin (arm repeat) or importin-b1 (HEAT repeat) show that the conserved hydrophobic residues located in this region align very well amongst these structures (Figure 5(a) and(b)). Helix A of HEAT repeats is relatively variable in sequence and in its degree of bending, whereas arm repeats are more regular. Thus, the N-terminal part of importin-b1 helix A and the H1 helices of b-catenin and PKP1 arm repeats are positioned differently. In arm repeats, a conserved glycine residue links H1 and H2. The structures of b-catenin and importin-a show that this glycine residue orients H2 at 908 relative to H1. Regular positioning of H1 within each repeat results in a relatively uniform spatial relationship between neighboring repeats. In contrast, only four of the nine PKP1 arm repeats have Gly at this position, which gives rise to a less regular relationship between H1 and H2, and between neighboring repeats in PKP1. For example, comparison of PKP1 repeat 4, which does not have Gly between H1 and H2, with the arm repeats of b-catenin reveals a significant difference in the H1
371 positions (Figure 6). This slightly alters the interrepeat packing: the rotation angle between R3 and R4 of PKP1 is about 108, whereas the corresponding angle in b-catenin is about 288. In addition to the repeat 5–6 insert, the PKP1 arm domain has two other significant loops. In repeat 4, a 13 amino acid residue loop that includes nine disordered residues connects H2 and H3. This loop affects the packing between R4 and R5, with a ˚ . The presence rotation of 308 and translation of 9 A of Gly385 at the end of H2, with the main-chain torsion angles fZC738, jZC428 seems to be responsible for the direction of the following flexible loop toward R5. Interactions between the terminal region of H2 and the beginning of H3 are enabled by the interaction between the side-chains of two aromatic residues, Phe383 in H2 and Phe403 in H3; Pro382 kinks H2 to position Phe383. In contrast to the alterations in packing caused by the repeat 4 loop, an eight amino acid residue loop that connects H2 and H3 of repeat 8 does not affect significantly the packing between adjacent repeats, because it protrudes outward from the main body of the repeat. Therefore, the presence of the variable length of loop between H2 and H3 may or may not affect the packing of two neighboring repeats, depending on its location and the particular
Figure 5. Comparison of repeat motif structures. (a) A comparison of PKP1 R3 with b-catenin R3. The backbones are overlaid on the left. The individual repeats are shown on the right, with the side-chains of the conserved consensus residues shown in ball-and-stick representation. (b) A comparison of PKP1 arm repeat 3 with importin-b HEAT repeat 3.
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Plakophilin 1 Armadillo Repeat Domain Structure
Figure 6. The effect of the H1–H2 turn on arm repeat structure. Comparison of PKP1 repeat 4, which does not contain the glycine residue found frequently in the H1– H2 turn, with b-catenin repeat 3, which has this glycine residue.
residues in its vicinity. Although the presence of inserts in the H2–H3 connection can alter the orientation of two neighboring repeats, the conserved inter-repeat hydrophobic interactions form a single continuous hydrophobic core and thereby maintain the basic architecture of the PKP1 arm domain. The most notable difference between PKP1 and the other known arm domain structures is the large insert between repeats 5 and 6, which functions as a wedge that bends the domain. When the N-terminal three repeats of PKP1 are aligned with the corresponding region of b-catenin, the C-terminal part of PKP1 is strongly bent relative to b-catenin near the insert (Figure 7). Although the insert was originally thought to be an arm repeat, it lacks the highly conserved consensus LxNLS sequence found in H3 of true arm repeats. Given that H3 is the most structurally uniform portion of arm repeats, it appears that the conservation of residues of H3 should be considered as a more important factor for sequence alignments of arm proteins or for detection of new arm repeat proteins. The highly bent PKP1 arm domain, which contrasts with the relatively straight b-catenin and importin-a structures, also shows that arm repeat proteins can have significant structural variation while maintaining the basic hydrophobic interactions that determine arm domain structures. Ligand binding by p120ctn-family arm proteins The side-chains of conserved Asn residues in the H3 LxNL motif are located on the external face of H3, where they project into the groove formed by the superhelical structure of the arm domain. This arrangement creates a regularly spaced array of
Figure 7. The effect of the insert on the overall structure of the PKP1 arm domain. Repeats 1–3 of PKP1 (red) were superimposed on repeats 1–3 of b-catenin (blue). A significant bend in the region of the PKP1 insert (circled) is apparent.
amide and carbonyl groups that form hydrogen bonds with the corresponding moieties of the backbone of importin-a and b-catenin ligands.4,12 The ability to satisfy the hydrogen bond potential of the peptide backbone allows protein ligands that are unstructured when free in solution to bind to their partner arm domains. The conservation of the H3 Asn residues in p120ctn family members suggests that these arm proteins bind to their protein ligands as extended peptides. In particular, the entire cytoplasmic domain of E-cadherin is unstructured in the absence of its binding partners,50 so it seems likely that the groove formed by the H3 helices serves as the cadherinbinding site in p120ctn. This is consistent with the observation that deleting the insert between repeats
373
Plakophilin 1 Armadillo Repeat Domain Structure
Figure 8. The electrostatic surface profile of PKP1. Blue and red represent regions of positive and negative electrostatic potential, respectively.
5 and 6 of p120ctn, which would not be expected to disrupt the continuous packing of arm repeats, has no effect on cadherin binding.44 The electrostatic surface profile of the PKP1 arm domain shows a positively charged patch in the superhelical groove (Figure 8). Since the binding of ligands to this groove is common to arm repeat proteins, the positive patch on this surface may represent the binding site for as yet unidentified PKP1 ligands. The positively charged residues that contribute to the basic character of the groove are conserved in p120 (Figure 3) and in b-catenin,2 but not in importin-a. b-Catenin has a positively charged groove that interacts with ligands that carry overall negative charge,4 whereas importin-a has a negatively charged groove that binds to positively charged NLS peptides.12 The juxtamembrane region in E-cadherin that binds to p120ctn features many acidic residues, some of which are essential for the interaction with p120ctn.25 Although there is a correlation between the overall charge of the ligands and a complementary character of its partner arm repeat domain, it is important to note that the structures of complexes between these arm domains and their ligands show that electrostatic complementarity is not the sole factor in forming a specific complex. The role of the positively charged surface of PKP1 must therefore await identification of partner proteins and determination of the structures of these complexes.
Materials and Methods Expression and purification of the arm repeat domain of PKP1 The region containing amino acid residues 237–704 of human PKP1 was cloned into a modified pGEX-KG bacterial expression vector by PCR, using a cDNA that was kindly provided by Dr M. Hatzfeld. The resulting construct encodes the full arm repeat region of PKP1, fused to the C terminus of glutathione-S-transferase (GST), by a tobacco etch virus (TEV) protease-cleavable linker. The fusion protein was overexpressed in the E. coli BL21 strain. Cells were grown for three hours at 28 8C after induction with 0.5 mM IPTG. Cells were harvested
by centrifugation and lysed by French press. Cleared cell lysates were incubated with glutathione-agarose beads for one hour at 4 8C. Beads were washed first with PBS containing 1 M NaCl and 5 mM DTT and then with cleavage buffer consisting of 25 mM Tris–HCl (pH 8.8), 75 mM NaCl, 3 mM DTT, 0.5 mM EDTA. TEV protease (one unit) was added for the cleavage of 3 mg of GSTfusion protein. The cleavage reaction was carried out at 4 8C for overnight. The cleaved protein was loaded onto a Mono Q column (Pharmacia) equilibrated with buffer A (25 mM ethanolamine (pH 9.5), 2 mM DTT, 0.5 mM EDTA). Protein was eluted by linear gradient from buffer A to buffer A C0.5 M NaCl. Peak fractions were pooled and loaded onto a Superdex S200 gel-filtration column (Pharmacia) equilibrated with buffer A containing 0.2 M NaCl. The purified proteins sizes at an apparent molecular mass of 45 kDa, corresponding to a monomer. Eluted protein was concentrated to 5 mg/ml for crystallization. Selenomethionyl protein was produced using metabolic inhibition of methionine synthesis. Cells were grown at 37 8C in M9 minimal medium to an absorbance at 600 nm (A600) of 0.7. At this point, the temperature was lowered to 28 8C and 100 mg/l each of D-lysine, D -phenylalanine and D -threonine, 50 mg/l of both D-isoleucine and D-valine, and 60 mg/l of D/L-selenomethionine were added. After 15 minutes, 0.5 mM IPTG was added. The cells were grown for an additional six hours at 28 8C. The cells were harvested and selenomethionyl protein was purified as described above. Crystallization and structure determination Native and selenomethionyl protein crystals were grown at 20 8C by the hanging-drop, vapor-diffusion method. Native protein crystals grew as thin plate clusters in 0.1 M Tris–HCl (pH 8.6), 10% (w/v) polyethylene glycol 3350, and 0.1 M potassium formate. A native data set was measured at ALS beamline 8.2.2 and processed and scaled with the HKL2000 package.51 Selenomethionyl protein crystals with dimensions of 10 mm!50 mm!20 mm were grown initially in 0.1 M Tris–HCl (pH 8.5), 9–10% polyethylene glycol 3350, 0.1 M potassium formate and 4 mM Tris(2-carboxyethyl)-phosphine (TCEP). Microseeding was used to produce larger crystals. SAD data were measured at ALS beamline 8.3.1. Data were processed using MOSFLM52 and SCALA53 (Table 2). The space group is C2221 with one molecule per asymmetric unit, corresponding to a solvent content of 44% (v/v). The program CNS54 was used to find and refine ten of a potential 14 selenium sites.
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Plakophilin 1 Armadillo Repeat Domain Structure
Table 2. Crystallographic statistics A. Data collection statistics Data set ˚) Wavelength (A Space group Unit cell parameters ˚) a (A ˚) b (A ˚) c (A ˚) Resolutiona (A Total observations Unique reflections Completenessa (%) I/s(I)a Rsyma,b B. Phasing statistics ˚) Resolutiona (A Phasing powerc Figure-of-merita C. Refinement statistics ˚) Resolution (A No. reflections working set (test set)d No. residues No. atoms Rcryst/Rfreee rmsd from ideality ˚) Bond lengths (A Bond angles (deg.) ˚ 2) Average B factor (A ˚ 2) Anisotropic B correction (A Ramachandran analysis Residues in most-favored region (%) Additionaly allowed (%) Generously allowed (%) Disallowed (%)
Native 0.9790 C2221
SAD 0.9796 C2221
53.35 131.98 142.04 50–2.8 (2.9–2.8) 57,896 13,094 92.1 (73.3) 11.6 (3.3) 0.055 (0.34)
53.56 132.30 143.18 50–3.2 (3.28–3.2) 67,102 8769 100.0 (99.9) 7.6 (2.6) 0.089 (0.30)
50–3.5 (3.6–3.5) 2.18/2.19 0.38 (0.25) 30–2.8 10,310 (1,142) 422 2930 0.25/0.32 0.0098 1.55 70.3 B11Z20.7/B22ZK40.2/B33Z19.5 340 (80.6) 72 (17.0) 8 (1.9) 2 (0.5)
a
Numbers P inPparentheses refer P toPthe highest resolution shell. Rsym Z h i jIi ðhÞK hIðhÞij= h i Ii ðhÞ, where Ii(h) is the ith measurement of reflection h and hI(h)i is the weighted mean of all measurements h. c Phasing powerZhjFHji/E, where hjFHji is the rms structure factor amplitude for the anomalous scatterers and E is the estimated lackof-closure error. Overall phasing powers are given separately for the C and K Friedel mates. d The P test set is a randomly selected subset of 10% of the total data and is not included in the refinement. P e RZ h jjFobs ðhÞjK jFcalc ðhÞjj= h jFobs ðhÞj. Rfree and Rcryst were calculated using the test set and working set reflections, respectively. b
Single-wavelength anomalous dispersion phase calculation (Table 2) followed by density modification was performed with CNS. The initial map clearly showed the arm repeats and made it possible to build a model with the program O.55 Simulated annealing, minimization and temperature factor refinement were carried out using CNS. Several rounds of minimization against the native data set and model rebuilding gave the final structure with RcrystZ0.25, and RfreeZ0.32. The final R values are higher than would be expected based on the quality of the final electron density maps at this resolution, which likely reflects the very significant anisotropy of the crystal (Table 2). Protein Data Bank accession code Coordinates and structure factors have been deposited in the Protein Data Bank with accession code ID 1XM9.
Acknowledgements We thank M. Hatzfeld for the PKP1 clone. Diffraction data were measured at the Advanced Light Source, Lawrence Berkeley National
Laboratory. This work was supported by grant GM56169 from the National Institutes of Health to W.I.W.
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7. Spink, K. E., Fridman, S. G. & Weis, W. I. (2001). Molecular mechanisms of b-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC/b-catenin complex. EMBO J. 20, 6203–6212. 8. Daniels, D. L. & Weis, W. I. (2002). ICAT inhibits b-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural modules. Mol. Cell, 10, 573–584. 9. Graham, T. A., Clements, W. K., Kimelman, D. & Xu, W. (2002). The crystal structure of the b-catenin/ICAT complex reveals the inhibitory mechanism of ICAT. Mol. Cell, 10, 563–571. 10. Ha, N.-C., Tonozuka, T., Stamos, J. L., Choi, H.-J. & Weis, W. I. (2004). Mechanism of phosphorylationdependent binding of APC to b-catenin and its role in b-catenin degradation. Mol. Cell, 15, 511–521. 11. Xing, Y., Clements, W. K., Le Trong, I., Hinde, T. R., Stenkamp, R., Kimelman, D. & Xu, W. (2004). Crystal structure of a b-catenin/APC complex reveals a critical role for APC phosphorylation in APC function. Mol. Cell, 15, 523–533. 12. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. (1998). Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin a. Cell, 94, 193–204. 13. Kobe, B. (1999). Autoinhibition by an internal nuclear localization signal revealsed by the crystal structure of mammalian importin a. Nature Struct. Biol. 6, 388–397. 14. Conti, E. & Kuriyan, J. (2000). Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha. Struct. Fold. Des. 8, 329–338. 15. Fontes, M. R. M., Teh, T. & Kobe, B. (2000). Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-a. J. Mol. Biol. 297, 1183–1194. 16. Fontes, M. R. M., Teh, T., Jans, D., Brinkworth, R. I. & Kobe, B. (2003). Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-a. J. Biol. Chem. 278, 27981–27987. 17. Matsuura, Y., Lange, A., Harreman, M. T., Corbett, A. H. & Stewart, M. (2003). Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import. EMBO J. 22, 5358–5369. 18. Anastasiadis, P. Z. & Reynonds, A. B. (2000). The p120 catenin family: complex roles in adhesion, signaling and cancer. J. Cell Sci. 113, 1319–1334. 19. Reynolds, A. B., Daniel, J., McCrea, P. D., Wheelock, M. J., Wu, J. & Zhang, Z. (1994). Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes. Mol. Cell. Biol. 14, 8333–8342. 20. Shibamoto, S., Hayakawa, M., Takeuchi, K., Hori, T., Miyazawa, K., Kitamura, N. et al. (1995). Association of p120, a tyrosine kinase substrate, with E-cadherin/ catenin complexes. J. Cell Biol. 128, 949–957. 21. Staddon, J. M., Smales, C., Schulze, C., Esch, F. S. & Rubin, L. L. (1995). p120, a p120-related protein (p100), and the cadherin/catenin complex. J. Cell Biol. 130, 369–381. 22. Myster, S. H., Cavallo, R., Anderson, C. T., Fox, D. T. & Peifer, M. (2003). Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component. J. Cell Biol. 160, 433–449. 23. Daniel, J. M. & Renynolds, A. B. (1995). The tyrosine
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kinase substrate p120cas binds directly to E-cadherin but not to the adenomatous polyposis coli protein or a-catenin. Mol. Cell. Biol. 15, 4819–4824. Ohkubo, T. & Ozawa, M. (1999). p120ctn binds to the membrane-proximal region of the E-cadherin cytoplasmic domain and is involved in modulation of adhesion activity. J. Biol. Chem. 274, 21409–21415. Thoreson, M. A., Anastasiadis, P. Z., Daniel, J. M., Ireton, R. C., Wheelock, M. J., Johnson, K. R. et al. (2000). Selective uncoupling of p120ctn from E-cadherin disrupts strong adhesion. J. Cell Biol. 148, 189–201. Yap, A. S., Niessen, C. M. & Gumbiner, B. M. (1998). The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn. J. Cell Biol. 141, 779–789. Davis, M. A., Ireton, R. C. & Reynolds, A. B. (2003). A core function for p120-catenin in cadherin turnover. J. Cell Biol. 163, 525–534. Xiao, K., Allison, D. F., Buckley, K. M., Kottke, M. D., Vincent, P. A., Faundez, V. & Kowalczyk, A. P. (2003). Cellular levels of p120 catenin function as a set point for cadherin expression levels in microvascular endothelial cells. J. Cell Biol. 163, 535–545. Chen, X., Kojima, S., Borisy, G. G. & Green, K. J. (2003). p120 catenin associates with kinesin and facilitates the transport of cadherin–catenin complexes to intercellular junctions. J. Cell Biol. 163, 547–557. Miranda, K. C., Joseph, S. R., Yap, A. S., Teasdale, R. D. & Stow, J. L. (2003). Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia. J. Biol. Chem. 278, 43480–43488. Anastasiadis, P. Z., Moon, S. Y., Thoreson, M. A., Mariner, D. J., Crawford, H. C., Zheng, Y. & Reynolds, A. B. (2000). Inhibition of RhoA by p120 catenin. Nature Cell Biol. 2, 637–644. Anastasiadis, P. Z. & Reynolds, A. B. (2001). Regulation of Rho ATPases by p120-catenin. Curr. Opin. Cell Biol. 13, 604–610. Magie, C. R., Pinto-Santini, D. & Parkhurst, S. M. (2002). Rho1 interacts wtih p120ctn and alpha-catenin, and regulates cadherin-based adherens junction components in Drosophila. Development, 129, 3771–3782. Kapprell, H. P., Owaribe, K. & Franke, W. W. (1988). Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia. J. Cell Biol. 106, 1679–1691. Mertens, C., Kuhn, C. & Franke, W. W. (1996). Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque. J. Cell Biol. 135, 1009–1025. Bonne, S., van Hengel, J., Nollet, F., Kools, P. & van Roy, F. (1999). Plakophilin-3, a novel Armadillo-like protein present in nuclei and desmosomes of epithelial cells. J. Cell Sci. 112, 2265–2276. Schmidt, A., Langbein, L., Pratzel, S., Rode, M., Rackwitz, H. R. & Franke, W. W. (1999). Plakophilin 3—a novel cell-type-specific desmosomal plaque protein. Differentiation, 64, 291–306. Hatzfeld, M. & Nachtsheim, C. (1996). Cloning and characterization of a new armadillo family member, p0071, associated with the junctional plaque: evidence for a subfamily of closely related proteins. J. Cell Sci. 109, 2767–2778. Smith, E. A. & Fuchs, E. (1998). Defining the interactions between intermediate filaments and desmosomes. J. Cell Biol. 141, 1229–1241.
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40. Kowalczyk, A. P., Hatzfeld, M., Bornslaeger, E. A., Kopp, D. S., Borgwardt, J. E., Corcoran, C. M. et al. (1999). The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. J. Biol. Chem. 274, 18145–18148. 41. Hatzfeld, M., Haffner, C., Schulze, K. & Vinzens, U. (2000). The function of plakophilin 1 in desmosome assembly and actin filament organization. J. Cell Biol. 149, 209–222. 42. Hofmann, I., Mertens, C., Brettel, M., Nimmrich, V., Schnolzer, M. & Herrmann, H. (2000). Interaction of plakophilins with desmoplakin and intermediate filament proteins: an invitro analysis. J. Cell Sci. 113, 2471–2483. 43. Hatfeld, M., Green, K. J. & Sauter, H. (2003). Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains. J. Cell Sci. 116, 1219–1233. 44. Roczniak-Ferguson, A. & Reynolds, A. B. (2003). Regulation of p120-catenin nucleocytoplasmic shuttling activity. J. Cell Sci. 116, 4201–4212. 45. Groves, M. R., Hanlon, N., Turowski, P., Hemmings, B. A. & Barford, D. (1999). The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell, 96, 99–110. 46. Cingolani, G., Petosa, C., Weis, K. & Muller, C. W. (1999). Structure of importin-b bound to the IBB domain of importin-a. Nature, 399, 221–229. 47. Chook, Y. M. & Blobel, G. (1999). Structure of the nuclear transport complex karyopherin-b2Ran.GppNHp. Nature, 399, 230–237.
48. Edwards, T. A., Pyle, S. E., Wharton, R. P. & Aggarwal, A. K. (2001). Structure of Pumilio reveals similarity between RNA and peptide binding motifs. Cell, 105, 281–289. 49. Wang, X., Zamore, P. D. & Hall, T. M. (2001). Crystal structure of a Pumilio homology domain. Mol. Cell, 7, 855–865. 50. Huber, A. H., Stewart, D. B., Laurents, D. V., Nelson, W. J. & Weis, W. I. (2001). The cadherin cytoplasmic domain is unstructured in the absence of b-catenin: a possible mechanism for regulating cadherin turnover. J. Biol. Chem. 276, 12301–12309. 51. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. 52. Leslie, A.G.W. (1993). Autoindexing of rotation diffracton images and parameter refinement. In Proceedings of the CCP4 Study Weekend: Data Collection and Processing, 29–30 January 1993 (Sawyer, L., Isaacs, N. & Bailey, S., eds.), pp. 44–51, SERC Daresbury Laboratory, Daresbury, UK. 53. Collaborative Computational Project No. 4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallog sect. D, 50, 760–763. 54. Bru¨nger, A. T., Adams, P. D., Clore, G. M., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S. et al. (1998). Crystallography and NMR system (CNS): a new software system for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905–921. 55. Jones, T. A. & Kjeldgaard, M. (1997). Electron-density map interpretation. Methods Enzymol. 277, 173–208.
Edited by R. Huber (Received 10 September 2004; received in revised form 15 November 2004; accepted 19 November 2004)
J. Mol. Biol. (2005) 346, 367–376
Structure of the Armadillo Repeat Domain of Plakophilin 1 Hee-Jung Choi and William I. Weis* Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine 299 Campus Drive West Stanford, CA 94305-5126 USA
The p120ctn subfamily of armadillo domain proteins has roles in modulating intercellular adhesion by cadherin-containing junctions. We have determined the crystal structure of the arm repeat domain from plakophilin-1 (PKP1), a member of the p120ctn subfamily that is found in desmosomes. The structure reveals that the domain has nine instead of the expected ten arm repeats. A sequence predicted to be an arm repeat is instead a large insert which serves as a wedge that produces a significant bend in the overall domain structure. Structure-based sequence alignments indicate that the nine repeats and large insert are common to this subfamily of armadillo proteins. A prominent basic patch on the surface of the protein may serve as a binding site for partners of these proteins. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: adherens junctions; desmosomes; armadillo proteins; plakophilins; p120ctn
Introduction Armadillo (arm) repeat proteins mediate protein– protein interactions in diverse cellular processes, including cell junction assembly, nuclear transport, and transcriptional activation. Proteins in this family, which includes the adherens junction component and transcription factor b-catenin, and the nuclear import protein importin-a, contain multiple copies (typically 7–13) of a 42 amino acid residue repeat motif.1 Each repeat consists of three a helices, designated H1, H2, and H3.2 Successive arm repeats pack together to form an elongated superhelical structure. Structural studies of b-catenin3–11 and importin-a12–17 have shown that many of their ligands bind in the groove formed by the superhelical structure of the arm repeat domain. Several different arm proteins are present in cadherin-containing intercellular junctions (Figure 1). In adherens junctions, b-catenin binds to the cytoplasmic domain of classical cadherins. This complex is in turn linked to the actin cytoskeleton by a-catenin. Plakoglobin, which is highly homologous to b-catenin, is found in both adherens junctions and desmosomes. Plakoglobin can replace b-catenin in adherens junctions, where it binds to cadherins and to a-catenin. In desmosomes, plakoglobin interacts with the desmosomal Abbreviations used: Arm, armadillo; PKP, plakophilin. E-mail address of the corresponding author: [email protected]
cadherins, desmogleins and desmocollins. The plakoglobin/desmosomal cadherin complex is connected to intermediate filaments through desmoplakin. The protein p120ctn and several other homologous proteins form a subgroup of junctional arm proteins whose sequences are distinct from those of b-catenin and plakoglobin.18 These proteins contain a central arm repeat domain predicted to have ten arm repeats, flanked by N and C-terminal regions of varying sizes. p120ctn associates with cadherin/ catenin complexes19–21 and appears to modulate adhesion.18,22 The arm repeat region of p120ctn binds to a juxtamembrane site in the cytoplasmic domain of cadherins distinct from that of b-catenin,23–25 thereby forming a ternary complex with b-catenin and E-cadherin. The precise role of p120ctn is unclear, but it may influence the ability of cadherins to form cell-surface dimers thought to be the functionally adhesive unit.24–26 Recent evidence indicates that p120ctn has a role in the trafficking of cadherins to developing adherens junctions and in cadherin turnover.27–30 p120ctn may serve in regulating cytoskeletal assembly at junctions by virtue of its interactions with Rho-family GTPases.31–33 Plakophilins (PKPs) are p120ctn relatives found in desmosomes. Several types of PKPs have been detected at different cellular locations or at different stages of development. PKP1 is expressed predominantly in the suprabasal layers of stratified and complex epithelia, 34 whereas PKP2 is expressed in all simple, complex and stratified
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
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Plakophilin 1 Armadillo Repeat Domain Structure
Figure 1. A diagram of adherens junctions and desmosomes. Armadillo repeat proteins are shown in colors, with blue representing b-catenin and plakoglobin, and orange/red representing p120ctn and plakophilin. In adherens junctions, p120ctn binds to the cytoplasmic domain of classical cadherins. Plakophilins are desmosomal proteins thought to participate in the linkage between the desmosomal cadherin–plakoglobin complex and desmoplakin.
epithelia as well as non-epithelial tissues such as myocardium and lymph node follicles.35 PKP3 is found at simple and almost all stratified epithelia except for hepatocytes and hepatocellular carcinoma cells and it is expressed uniformly in epidermal layers.36,37 PKP4, which is known also as p0071, is expressed in endothelial cells and it is unique amongst the p120ctn subfamily, in that it is found in both adherens junctions and desmosomes.38 All PKPs have been found in the nucleus as well as at cell junctions, but the significance of this observation remains unclear. The arm repeat domains of b-catenin, plakoglobin, and p120ctn mediate the interactions of these proteins with many of their binding partners. In contrast, the N-terminal head domain of PKPs appears to be responsible for the interaction with their desmosomal partners, including desmoplakin, desmocollins, desmogleins, and intermediate filaments.39–42 Indeed, the arm domain of p0071 is required for adherens junction localization, whereas its N-terminal head region is responsible for desmosomal targeting.43 The arm domain of PKP1 has been observed to associate with actin filaments, suggesting that PKP1 may be involved in the regulation of the actin cytoskeleton.41 However, yeast two-hybrid analysis showed that the arm domain of PKP1 did not interact with actin monomer,41 and cosedimentation assays using purified F-actin filaments showed that PKP1 did not associate with actin filaments in vitro (our unpublished results), implying that other PKP1 interacts with the actin cytoskeleton through other actin-binding proteins. As more information emerges about the modulatory roles of p120ctn and its relatives in cell adhesion, structural data for this distinct subgroup of arm proteins will be important for understanding the molecular basis of their interactions. Here, we present the first arm domain structure from the p120ctn family, that of PKP1. The structure shows significant differences with previously solved arm
domain structures, and demonstrates that this subfamily contains nine rather than the predicted ten arm repeats. A prominent basic groove formed by the structure likely represents a ligand-binding surface.
Results and Discussion Overall structure Structure-based sequence alignments of PKP1 indicate that residues 237–704 correspond to the arm repeat domain. This region was expressed in Escherichia coli, purified, and crystallized. The ˚ resolution by singlestructure was solved at 2.8 A wavelength anomalous dispersion phasing of selenomethionyl protein. The final model comprises amino acid residues 243–387, 396–480, and 509–700. The PKP1 arm region structure reveals a single domain with nine repeats (Figure 2). In contrast to other known arm repeat structures, the PKP1 domain is sickle-shaped rather than straight, with ˚ . The diameter of a crossan overall length of 95 A ˚ . A long, section through the repeats is about 32 A flexible insert containing residues 464–524 is present between the fifth and sixth arm repeats. This region does not disrupt the continuous packing of the repeats, but is the site of a major bend in the arm domain. The individual PKP1 arm repeats are very similar to those found in other arm domain structures. Each repeat is composed of three helices, H1, H2 and H3, with linkers of highly variable length between H2 and H3. Highly conserved residues mediate the packing interactions between neighboring repeats and within single repeats (Figure 3). Most conserved hydrophobic residues in H3 are involved in intra-repeat packing or hydrophobic contacts with the next repeat. In particular, H3 has a very highly conserved LxNL sequence motif near its C
Plakophilin 1 Armadillo Repeat Domain Structure
Figure 2. The overall structure of the PKP1 arm repeat domain.
terminus. The two leucine residues participate in intra-repeat hydrophobic interactions with conserved hydrophobic residues in the middle of H2 and H1, respectively (Figure 3). Other hydrophobic residues conserved in H2 and H3 mediate interrepeat packing interactions to form a contiguous hydrophobic core throughout the domain. Among
369 three conserved Leu residues in H2, the first and the third interact with the hydrophobic residues of the next repeat, and the second, which is located at the opposite side of helix H2, interacts with the previous repeat. All repeats except R6 have similar secondary structural elements. R6, which is followed by the insert, does not have an H1 (Figures 2–4). Instead, the space that would be occupied by H1 in a standard arm repeat is taken by a part of the repeat 5–6 insert, which forms contacts distinct from those normally made by H1 (see below). Nonetheless, H2 and H3 of R6 align well with the corresponding regions of other repeats. Even though the arm repeats of PKP1 are similar, there are some variations in the position of H1 relative to H2 and H3. The angles between H1 and H2 vary from 558 to 1058, as compared with the smaller variation of 358 to 558 found in the H2–H3 angles (Table 1). The variability in the position of H1 relative to H2 and H3 correlates with sequence variations. Repeats 2, 3, 5 and 7 have at least one glycine residue between H1 and H2, which makes a sharp bend between H1 and H2 of about 708 to 908. In the repeats lacking this glycine residue, the angles between H1 and H2 are more variable, ranging from 578 to 1008. Because H1 is connected to H3 of the previous repeat by a very short turn composed of one to three amino acid residues, the position of H1 affects the relative location of the
Figure 3. The structure-based sequence alignment of PKP1 and p120ctn arm domains. PKP1 residues comprising H1, H2, and H3 in each repeat are boxed; the green box indicates the 310 helix in the insert. Italicized PKP1 residues are disordered in the structure. Conserved residues that define the arm consensus motif are highlighted in yellow, and the consensus is shown at the bottom of the figure. In the consensus, the single-letter code is listed if the residue is present in at least two-thirds of the repeats. Consensus positions with large or small hydrophobic residues are shown as filled or open boxes, respectively, and those with either small or large hydrophobic residues as half-filled boxes. The C sign indicates a positively charged residue. For comparison, the consensus arm repeat motif derived from the b-catenin structure2 is indicated at the bottom. Conserved residues that mediate contacts between the insert and repeats 5–7 are highlighted in blue.
370
Plakophilin 1 Armadillo Repeat Domain Structure
An insert replaces a predicted arm repeat
Figure 4. Interactions of the repeat 5–6 insert. Repeats 5–7 are colored as in Figure 2, and H1, H2 and H3 in each repeat are indicated. The insert is shown with a grey backbone, except for the 310 helix containing Leu522, which is indicated by the dark grey backbone. Key residues that mediate the interactions are shown in balland-stick representation. Carbon, nitrogen, oxygen, and sulfur atoms are shown as yellow, blue, red, and green spheres, respectively. Salt bridges between Glu468 and Arg420 and Arg424 are shown as broken black lines.
previous repeat. This can be seen in the rotation and translation between neighboring repeats of PKP1, ˚ to 9.5 A ˚, which vary from 108 to 308 and from 8.5 A respectively. Despite the variation in the relative positions of H1 amongst the repeats, the packing interactions mediated by the conserved hydrophobic residues of H1 are preserved throughout the structure.
Table 1. Inter-helix angles within PKP-1 arm repeats Helix angle (8) Repeat R1 R2 R3 R4 R5 R6 R7 R8 R9 AverageGSD
H1–H2
H2–H3
N/A 78.5 67.9 55.6 79.5 N/A 93.6 58.3 104.7 76.9G18.0
40.8 35.6 32.7 47.1 46.1 35.4 40.1 37.6 54.5 41.1G7.0
On the basis of the sequence alignments of b-catenin and p120ctn-family proteins,18 part of the 61 amino acid residue insert between repeats 5 and 6 was expected to form an arm repeat. Instead, this region starts as two a-helices that protrude outward from the main body of the structure, and are interrupted by a conserved proline residue. These helices are followed by a flexible loop of 28 residues (italics in Figure 3), and the insert ends as a 310 helix of seven residues. The central portion of the insert is not visible in the electron density map and therefore unstructured, but the beginning and ends of the insert are structured and stabilized by interactions with conserved residues in repeats 5 and 7 (Figure 4). Glu468, which is located at the beginning of the insert and is highly conserved in all p120ctn family members, forms salt bridges with the conserved, positively charged residues Arg420 and Arg424 in R5 (Figure 4). Leu465, Val469, and Tyr473 at the beginning of the insert, and Leu522 and Tyr523 at the end, pack with Met561 and Met565 of R7 (Figures 3 and 4). Although most of the insert sequence is not conserved in p120ctn family members, the residues of the loop that interact with the main body of the protein are highly conserved, implying that other p120ctn homologs have a similar structure in this region. Therefore, we predict that all members of the p120ctn subfamily of arm proteins will have nine instead of ten arm repeats, and the numbering of the repeats used here reflects this fact. Evidence for an insert rather than a repeat at residues 589 to 661 of p120ctn comes also from the observation that deletion of this sequence does not affect binding to E-cadherin, whereas the flanking arm repeats are required for this interaction.44 Comparisons with other structures containing a-helical repeats The structures of two other arm repeat domain structures, b-catenin 2 and importin-a 12,13 are known, as are several other a-solenoid structures composed of helical repeats such as HEAT45–47 and Pumilio motifs.48,49 None of these structures has a repeat-length insert between neighboring repeats such as that present in the PKP1 arm domain. It is interesting to note that the large bend in PKP1 that arises from this insert prevented determination of the structure using molecular replacement phasing from known arm domain models. Even though the overall structures of arm domain and HEAT repeat-containing proteins are somewhat different, it has been noted that they likely have a common origin.1 The main feature that distinguishes these types of repeat is that arm repeats contain three helices, H1, H2 and H3, whereas HEAT repeats have just two helices, denoted A and B.45,46 Helix A of the HEAT repeat is usually bent, and corresponds to H1 and H2 of an arm repeat, and helix B corresponds to arm repeat
Plakophilin 1 Armadillo Repeat Domain Structure
H3. The arm and HEAT repeats share the conserved hydrophobic residues that mediate inter-repeat and intra-repeat interactions. Structural alignment between H2 and H3 of PKP1 and the corresponding regions of b-catenin (arm repeat) or importin-b1 (HEAT repeat) show that the conserved hydrophobic residues located in this region align very well amongst these structures (Figure 5(a) and(b)). Helix A of HEAT repeats is relatively variable in sequence and in its degree of bending, whereas arm repeats are more regular. Thus, the N-terminal part of importin-b1 helix A and the H1 helices of b-catenin and PKP1 arm repeats are positioned differently. In arm repeats, a conserved glycine residue links H1 and H2. The structures of b-catenin and importin-a show that this glycine residue orients H2 at 908 relative to H1. Regular positioning of H1 within each repeat results in a relatively uniform spatial relationship between neighboring repeats. In contrast, only four of the nine PKP1 arm repeats have Gly at this position, which gives rise to a less regular relationship between H1 and H2, and between neighboring repeats in PKP1. For example, comparison of PKP1 repeat 4, which does not have Gly between H1 and H2, with the arm repeats of b-catenin reveals a significant difference in the H1
371 positions (Figure 6). This slightly alters the interrepeat packing: the rotation angle between R3 and R4 of PKP1 is about 108, whereas the corresponding angle in b-catenin is about 288. In addition to the repeat 5–6 insert, the PKP1 arm domain has two other significant loops. In repeat 4, a 13 amino acid residue loop that includes nine disordered residues connects H2 and H3. This loop affects the packing between R4 and R5, with a ˚ . The presence rotation of 308 and translation of 9 A of Gly385 at the end of H2, with the main-chain torsion angles fZC738, jZC428 seems to be responsible for the direction of the following flexible loop toward R5. Interactions between the terminal region of H2 and the beginning of H3 are enabled by the interaction between the side-chains of two aromatic residues, Phe383 in H2 and Phe403 in H3; Pro382 kinks H2 to position Phe383. In contrast to the alterations in packing caused by the repeat 4 loop, an eight amino acid residue loop that connects H2 and H3 of repeat 8 does not affect significantly the packing between adjacent repeats, because it protrudes outward from the main body of the repeat. Therefore, the presence of the variable length of loop between H2 and H3 may or may not affect the packing of two neighboring repeats, depending on its location and the particular
Figure 5. Comparison of repeat motif structures. (a) A comparison of PKP1 R3 with b-catenin R3. The backbones are overlaid on the left. The individual repeats are shown on the right, with the side-chains of the conserved consensus residues shown in ball-and-stick representation. (b) A comparison of PKP1 arm repeat 3 with importin-b HEAT repeat 3.
372
Plakophilin 1 Armadillo Repeat Domain Structure
Figure 6. The effect of the H1–H2 turn on arm repeat structure. Comparison of PKP1 repeat 4, which does not contain the glycine residue found frequently in the H1– H2 turn, with b-catenin repeat 3, which has this glycine residue.
residues in its vicinity. Although the presence of inserts in the H2–H3 connection can alter the orientation of two neighboring repeats, the conserved inter-repeat hydrophobic interactions form a single continuous hydrophobic core and thereby maintain the basic architecture of the PKP1 arm domain. The most notable difference between PKP1 and the other known arm domain structures is the large insert between repeats 5 and 6, which functions as a wedge that bends the domain. When the N-terminal three repeats of PKP1 are aligned with the corresponding region of b-catenin, the C-terminal part of PKP1 is strongly bent relative to b-catenin near the insert (Figure 7). Although the insert was originally thought to be an arm repeat, it lacks the highly conserved consensus LxNLS sequence found in H3 of true arm repeats. Given that H3 is the most structurally uniform portion of arm repeats, it appears that the conservation of residues of H3 should be considered as a more important factor for sequence alignments of arm proteins or for detection of new arm repeat proteins. The highly bent PKP1 arm domain, which contrasts with the relatively straight b-catenin and importin-a structures, also shows that arm repeat proteins can have significant structural variation while maintaining the basic hydrophobic interactions that determine arm domain structures. Ligand binding by p120ctn-family arm proteins The side-chains of conserved Asn residues in the H3 LxNL motif are located on the external face of H3, where they project into the groove formed by the superhelical structure of the arm domain. This arrangement creates a regularly spaced array of
Figure 7. The effect of the insert on the overall structure of the PKP1 arm domain. Repeats 1–3 of PKP1 (red) were superimposed on repeats 1–3 of b-catenin (blue). A significant bend in the region of the PKP1 insert (circled) is apparent.
amide and carbonyl groups that form hydrogen bonds with the corresponding moieties of the backbone of importin-a and b-catenin ligands.4,12 The ability to satisfy the hydrogen bond potential of the peptide backbone allows protein ligands that are unstructured when free in solution to bind to their partner arm domains. The conservation of the H3 Asn residues in p120ctn family members suggests that these arm proteins bind to their protein ligands as extended peptides. In particular, the entire cytoplasmic domain of E-cadherin is unstructured in the absence of its binding partners,50 so it seems likely that the groove formed by the H3 helices serves as the cadherinbinding site in p120ctn. This is consistent with the observation that deleting the insert between repeats
373
Plakophilin 1 Armadillo Repeat Domain Structure
Figure 8. The electrostatic surface profile of PKP1. Blue and red represent regions of positive and negative electrostatic potential, respectively.
5 and 6 of p120ctn, which would not be expected to disrupt the continuous packing of arm repeats, has no effect on cadherin binding.44 The electrostatic surface profile of the PKP1 arm domain shows a positively charged patch in the superhelical groove (Figure 8). Since the binding of ligands to this groove is common to arm repeat proteins, the positive patch on this surface may represent the binding site for as yet unidentified PKP1 ligands. The positively charged residues that contribute to the basic character of the groove are conserved in p120 (Figure 3) and in b-catenin,2 but not in importin-a. b-Catenin has a positively charged groove that interacts with ligands that carry overall negative charge,4 whereas importin-a has a negatively charged groove that binds to positively charged NLS peptides.12 The juxtamembrane region in E-cadherin that binds to p120ctn features many acidic residues, some of which are essential for the interaction with p120ctn.25 Although there is a correlation between the overall charge of the ligands and a complementary character of its partner arm repeat domain, it is important to note that the structures of complexes between these arm domains and their ligands show that electrostatic complementarity is not the sole factor in forming a specific complex. The role of the positively charged surface of PKP1 must therefore await identification of partner proteins and determination of the structures of these complexes.
Materials and Methods Expression and purification of the arm repeat domain of PKP1 The region containing amino acid residues 237–704 of human PKP1 was cloned into a modified pGEX-KG bacterial expression vector by PCR, using a cDNA that was kindly provided by Dr M. Hatzfeld. The resulting construct encodes the full arm repeat region of PKP1, fused to the C terminus of glutathione-S-transferase (GST), by a tobacco etch virus (TEV) protease-cleavable linker. The fusion protein was overexpressed in the E. coli BL21 strain. Cells were grown for three hours at 28 8C after induction with 0.5 mM IPTG. Cells were harvested
by centrifugation and lysed by French press. Cleared cell lysates were incubated with glutathione-agarose beads for one hour at 4 8C. Beads were washed first with PBS containing 1 M NaCl and 5 mM DTT and then with cleavage buffer consisting of 25 mM Tris–HCl (pH 8.8), 75 mM NaCl, 3 mM DTT, 0.5 mM EDTA. TEV protease (one unit) was added for the cleavage of 3 mg of GSTfusion protein. The cleavage reaction was carried out at 4 8C for overnight. The cleaved protein was loaded onto a Mono Q column (Pharmacia) equilibrated with buffer A (25 mM ethanolamine (pH 9.5), 2 mM DTT, 0.5 mM EDTA). Protein was eluted by linear gradient from buffer A to buffer A C0.5 M NaCl. Peak fractions were pooled and loaded onto a Superdex S200 gel-filtration column (Pharmacia) equilibrated with buffer A containing 0.2 M NaCl. The purified proteins sizes at an apparent molecular mass of 45 kDa, corresponding to a monomer. Eluted protein was concentrated to 5 mg/ml for crystallization. Selenomethionyl protein was produced using metabolic inhibition of methionine synthesis. Cells were grown at 37 8C in M9 minimal medium to an absorbance at 600 nm (A600) of 0.7. At this point, the temperature was lowered to 28 8C and 100 mg/l each of D-lysine, D -phenylalanine and D -threonine, 50 mg/l of both D-isoleucine and D-valine, and 60 mg/l of D/L-selenomethionine were added. After 15 minutes, 0.5 mM IPTG was added. The cells were grown for an additional six hours at 28 8C. The cells were harvested and selenomethionyl protein was purified as described above. Crystallization and structure determination Native and selenomethionyl protein crystals were grown at 20 8C by the hanging-drop, vapor-diffusion method. Native protein crystals grew as thin plate clusters in 0.1 M Tris–HCl (pH 8.6), 10% (w/v) polyethylene glycol 3350, and 0.1 M potassium formate. A native data set was measured at ALS beamline 8.2.2 and processed and scaled with the HKL2000 package.51 Selenomethionyl protein crystals with dimensions of 10 mm!50 mm!20 mm were grown initially in 0.1 M Tris–HCl (pH 8.5), 9–10% polyethylene glycol 3350, 0.1 M potassium formate and 4 mM Tris(2-carboxyethyl)-phosphine (TCEP). Microseeding was used to produce larger crystals. SAD data were measured at ALS beamline 8.3.1. Data were processed using MOSFLM52 and SCALA53 (Table 2). The space group is C2221 with one molecule per asymmetric unit, corresponding to a solvent content of 44% (v/v). The program CNS54 was used to find and refine ten of a potential 14 selenium sites.
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Plakophilin 1 Armadillo Repeat Domain Structure
Table 2. Crystallographic statistics A. Data collection statistics Data set ˚) Wavelength (A Space group Unit cell parameters ˚) a (A ˚) b (A ˚) c (A ˚) Resolutiona (A Total observations Unique reflections Completenessa (%) I/s(I)a Rsyma,b B. Phasing statistics ˚) Resolutiona (A Phasing powerc Figure-of-merita C. Refinement statistics ˚) Resolution (A No. reflections working set (test set)d No. residues No. atoms Rcryst/Rfreee rmsd from ideality ˚) Bond lengths (A Bond angles (deg.) ˚ 2) Average B factor (A ˚ 2) Anisotropic B correction (A Ramachandran analysis Residues in most-favored region (%) Additionaly allowed (%) Generously allowed (%) Disallowed (%)
Native 0.9790 C2221
SAD 0.9796 C2221
53.35 131.98 142.04 50–2.8 (2.9–2.8) 57,896 13,094 92.1 (73.3) 11.6 (3.3) 0.055 (0.34)
53.56 132.30 143.18 50–3.2 (3.28–3.2) 67,102 8769 100.0 (99.9) 7.6 (2.6) 0.089 (0.30)
50–3.5 (3.6–3.5) 2.18/2.19 0.38 (0.25) 30–2.8 10,310 (1,142) 422 2930 0.25/0.32 0.0098 1.55 70.3 B11Z20.7/B22ZK40.2/B33Z19.5 340 (80.6) 72 (17.0) 8 (1.9) 2 (0.5)
a
Numbers P inPparentheses refer P toPthe highest resolution shell. Rsym Z h i jIi ðhÞK hIðhÞij= h i Ii ðhÞ, where Ii(h) is the ith measurement of reflection h and hI(h)i is the weighted mean of all measurements h. c Phasing powerZhjFHji/E, where hjFHji is the rms structure factor amplitude for the anomalous scatterers and E is the estimated lackof-closure error. Overall phasing powers are given separately for the C and K Friedel mates. d The P test set is a randomly selected subset of 10% of the total data and is not included in the refinement. P e RZ h jjFobs ðhÞjK jFcalc ðhÞjj= h jFobs ðhÞj. Rfree and Rcryst were calculated using the test set and working set reflections, respectively. b
Single-wavelength anomalous dispersion phase calculation (Table 2) followed by density modification was performed with CNS. The initial map clearly showed the arm repeats and made it possible to build a model with the program O.55 Simulated annealing, minimization and temperature factor refinement were carried out using CNS. Several rounds of minimization against the native data set and model rebuilding gave the final structure with RcrystZ0.25, and RfreeZ0.32. The final R values are higher than would be expected based on the quality of the final electron density maps at this resolution, which likely reflects the very significant anisotropy of the crystal (Table 2). Protein Data Bank accession code Coordinates and structure factors have been deposited in the Protein Data Bank with accession code ID 1XM9.
Acknowledgements We thank M. Hatzfeld for the PKP1 clone. Diffraction data were measured at the Advanced Light Source, Lawrence Berkeley National
Laboratory. This work was supported by grant GM56169 from the National Institutes of Health to W.I.W.
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Edited by R. Huber (Received 10 September 2004; received in revised form 15 November 2004; accepted 19 November 2004)