Structure of the C-cadherin ectodomain and implications for the mechanism of cell adhesion

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1 Structure of the C-cadherin ectodomain and implications for the mechanism of cell adhesion Titus J. Boggon, John Murray, Sophie Chappuis-Flament, Ellen Wong, Barry M. Gumbiner, and Lawrence Shapiro Ref. # Supporting Material Supporting Methods and Materials Protein Expression and Purification The C-cadherin extracellular domain (mature protein residues 1-542, following the native signal sequence and pre-domain) was expressed as a C-terminal 6-His tag fusion in Chinese Hamster Ovary (CHO) cells, and purified as previously described (18). Yields were typically ~1mg per liter of conditioned media, and purity was judged to be greater than 95% by gel electrophoresis. Protein Crystallization, Data Collection, Solution and Refinement Crystallizations were performed using the hanging drop method with protein at an initial concentration of 10 mg/ml in 10 mm Tris (ph 8.0), 5 mm CaCl 2 and 0.15 M NaCl (0.6 µl), mixed with well solution (0.6 µl) of 5-8 % PEG 4K, 10 mm Na-cacodylate (ph 5.0), 0.3 M NaCl, 30 mm MnCl 2 and 5 mm CaCl 2. Crystals (dimensions ~ mm) formed in well conditions between 5% and 8% PEG 4K at 20 C. Substantial Boggon et al. Page 1

2 non-isomorphism was a problem, and although all crystals tested were in space group C2, and contained one C-cadherin molecule per asymmetric unit, axis lengths ranged between 123 and 128 Å for a, 72 and 76 Å for b, and 128 and 132 Å for c, with β ranging between 104 and 106. Data were integrated and merged using the HKL software suite (S1). The best data (to 3.08 Å, Table 1) were collected from a native crystal in space group C2 with a=127.2 Å, b=75.1 Å, c=129.8 Å, β=105.5, at beamline X4A of the National Synchrotron Light Source (NSLS). Diffraction was highly anisotropic with the B-factor decrease along the worst diffracting axis 1.7 times that of the best diffracting orthogonal axis. Crystals were flash-cooled to 100K in stabilization buffer supplemented with 30% glycerol. Screening for heavy atom derivatives in these crystals to provide phasing proved nearly impossible, with severe non-isomorphism problems hampering acquisition of useful phasing information from the one derivative we found (YbCl 3 ). A MAD data collection was not possible as substantial deterioration of high-resolution data occurred in these small crystals. Typically, diffraction beyond 6Å resolution was completely absent after about 200 of data collection. Nonetheless, the clear single ytterbium site seen in the highest resolution data set collected for this derivative (to 6Å resolution) could be used to corroborate the structure in that it is bound in a single calcium binding site between domains 3 and 4 (Fig. S2B). The ytterbium data set integrated 289 of data from 20 to 6 Å resolution. The overall R sym was with an overall completeness of 96.6 %. A single clear molecular replacement solution for domains EC1 and EC2 was found using a two domain E-cadherin structure (PDB code: 1EDH) as a search model against the 3.08Å dataset using the program AMORE (36). Following rigid body refinement in X-PLOR (S2) the model was subjected to a single round of simulated Boggon et al. Page 2

3 annealing in CNS (S3). The program PRISM (D. A. Agard) was used to find areas of higher electron density. These correlated well with 3f obs -2f calc electron density maps made using phases from the two-domain model. A C α chain model of domain EC3 using coordinates from N-cadherin domain EC2 (PDB code: 1NCJ) was placed into the density in 20 different orientations using the program O (S4), and each model was optimized using rigid body refinement in X-PLOR with a target function based on correlation of squared normalized structure factors (E2E2). 19 of the 20 attempts deleteriously affected the correlation statistics compared to the two-domain model. One attempt to place domain EC3 substantially improved the correlation statistics. The resulting three-domain model was then subjected to rigid body refinement in X-PLOR and simulated annealing in CNS, yielding an R cryst of 37.9 % and R free of 46.1%. A number of rounds of refinement were performed using the program CNS on the three-domain model until the R cryst was 31.5% and the R free was 38.6%. Rounds of refinement followed a protocol of simulated annealing, conjugate gradient minimization, and restrained, grouped B-factor refinement, against the native data set to 3.08Å resolution using a standard crystallographic residual target and a 3σ data cutoff on F. Clear density was now visible and the protein model for domains EC4 and EC5 was built into areas with clear 2σ 3f obs - 2f calc and 3σ f obs -f calc density. Each piece of new model was added to the refined model individually, subjected to a round of refinement, and only kept if the R factor statistics improved. Water molecules and N-acetylglucosamine residues were added at a late stage in the refinement. One other intermolecular contact is observed in the crystals, in addition to the interfaces described in the main text. This is contact between domains EC3 and EC4 of symmetry-related protomers appears superficial and buries a surface area of 1400Å 2 ; we attribute this contact to crystal packing interactions. The final model had a crystallographic statistics, R cryst = 24.3% and R free = 27.6%. The final model had Boggon et al. Page 3

4 540 amino acid residues, 38 water molecules, 12 calcium atoms, 12 O-linked and 3 N- linked N-acetylglucosamine residues. There are no Ramachandran angle outliers, and the model geometry, as judged by the program PROCHECK (37), is better than that of the average of structures of similar resolution deposited in the Protein Data Bank. Boggon et al. Page 4

5 Supporting Text Structure The five EC domains are of similar fold (Fig. S1), with root-mean-square distances between equivalent alpha carbon atoms ranging between 1.6 and 2.0 Å (Table S1). Nonetheless, substantial differences among these domains are evident, with the terminal EC1 and EC5 domains being most unique. Of greatest note, a small feature previously termed a quasi-β-helix is found only in the EC1 domain, and sequence analysis suggests that this feature is conserved in the EC1 domains of type I classical cadherins, desmocollins, and desmogleins, but not in type II cadherins or protocadherins. Furthermore, a short β-stand, termed A*, preceding the A strand, is seen in all domains apart from EC1 (Fig. S2). EC5 is unique in that it includes two conserved disulfide bonds, Cys 448 Cys 532 and Cys 530 Cys 539, which link strands A to G, and G to the C-terminal tail, respectively. Interdomain linkages involve a rotation of ~120 between successive domains (EC1/EC2: 128 ; EC2/EC3: 106 ; EC3/EC4: 137 ; and EC4/EC5: 119 ), and translations of 50±2 Å for each successive domain pair. The angle of skew is similar between successive domains EC1/EC2, EC2 /EC3, and EC4/EC5, however, the skew angle at the EC3/EC4 interface is almost 180 different from the others (Fig. S2, and Table S2). This difference may arise due to differences in calcium binding at this particular interdomain interface (Fig. S3). The EC1/EC2, EC2/EC3, and EC4/EC5 calcium ligation schemes are almost identical, with corresponding residues in all these regions creating similar calcium binding sites. The EC3/EC4 calcium binding site, however, is distinct: in EC3/EC4 the side chain carboxyl from Gln 365 ligates Ca3, whereas this position is occupied by a main chain carbonyl from the start of β-strand C in all other interdomain Boggon et al. Page 5

6 regions. Gln 365, near the apex of the EC4 domain, is conserved in all classical cadherins (Fig. S4), suggesting a potential biological role. Strand dimer C-cadherin compared to other cadherin structures Comparisons between the C- and N-cadherin structures reveal substantial rotational freedom in the strand dimer interface deriving from variability in the χ1 and χ2 side chain angles of Trp 2, and the main chain Ramachandran angles of residues in the A strand, particularly those of Val 3 and Ile 4 (see Table S4). It should be noted that the N-terminal sequences of the first seven amino acids (DWVIPPI) of N, E, and C cadherins are identical, and all classical cadherins are either identical, or differ by at most two conservative substitutions (8). Furthermore, the two conserved proline residues at positions 5 and 6 may define a firm structural base from which rotations of the N- terminus can take place without affecting the remainder if the EC1 domain. Proline residues are highly restricted in their allowed ranges of Ramachandran angles, and notably almost all high-resolution cadherin structures adopt similar φ and ψ angles at these residues (19 out of 20 of these prolines have φ = 65 ±15 and ψ = 150 ±25 ). The strand dimer interaction has not been observed in all cadherin structures determined to date (Table S5). This may suggest the relative weakness of this interface, as has been indicated for the adhesive interactions of cadherins and cell adhesion proteins generally. We note Trp 2, the central and conserved ligand of this interface is a hydrophobic core residue that, unusually, has been found to be disordered in solvent leaving an exposed hydrophobic pocket in several of these structures. This unusual behavior is likely indicative of its function as an extrudable ligand. It is also of note that Boggon et al. Page 6

7 the constructs of both two-domain E-cadherin crystal structures to date (10, 17) include additional large residues at their N-termini, which might affect the behavior of their N- termini. Cis interaction Previously proposed cis interfaces Two other interfaces have previously been proposed to mediate cis interactions of cadherins. One of these is the strand dimer (9, 15), and mutagenesis data suggests that the strand dimer can function in cis dimerization (16, 20, 22), as well as trans adhesion as we suggest here. The other proposed cis interface, identified in the crystal structure of a 2-domain fragment from E-cadherin (10), is mediated by a total of two direct hydrogen bonds, and four water-mediated hydrogen bonds. We think that this interface is not biophysically tenable, and therefore is unlikely to represent a biologically relevant interaction. Implications Surface force measurements Surface force measurements have been performed on the C-cadherin ectodomain (31), identically the same molecule for the present crystal structure is reported. These experiments show the onset of steric repulsion between a cadherin monolayer and a bare Boggon et al. Page 7

8 lipid bilayer at ~230 Å, consistent with the estimate of 220 Å length of a cadherin ectodomain by electron micrographs (12, 13), and the head-to-tail length of the curved C-cadherin ectodomain found in the crystal of ~210 Å. We note that two residues linking the C-terminus to the transmembrane region and the associated 6-His tag are not seen in the crystal structure. It is possible that these features contribute to the measurements in the surface force studies, or that the curve of the molecule could vary. Unbinding between C-cadherin coated membrane surfaces present a complex picture of adhesion, which was interpreted to represent interaction between multiple cadherin domains (31). Specificity The conservation among classical cadherins of the core residues that define the adhesive interaction suggest a simple mechanism for cross-reactivity between different cadherins, and suggest that gradations of affinities among these cadherins could be defined by residue changes in limited regions surrounding the strand dimer interface. In C-cadherin, the side chain nitrogen of Lys 8 in the A strand forms a hydrogen bond to Gln 23 in the partner B strand (Table S3). These residues are different in different classical cadherins. Furthermore, the side chain of Trp 59 in the E strand of C-cadherin, which packs between the side chains of residues 20 and 23, is variable in different cadherins, and may also play a role in determining specificity. It is possible that there are other determinants of cadherin binding specificity outside of the strand dimer region. Additionally, the degree of adhesive specificity required for classical cadherin function is still an open question. For example, N-cadherin deficient mice die in midgestation, yet this phenotype is partially rescued with equal efficiency by ectopic Boggon et al. Page 8

9 expression of either N- or E-cadherin under the control of a heterologous promoter (S5). Most cells in vivo express a number of different cadherins (3, S6), and a large body of evidence suggests that substantial cross-interaction among different cadherins is likely an important aspect of their biological function (6). Boggon et al. Page 9

10 Supporting Figures Supporting Figure 1. All five EC domains from C-cadherin viewed in the same orientation. The side chain of Trp 2 is shown, calcium ions are depicted as green spheres, disulfides are yellow, O-linked sugars are red, and N-linked sugars are blue. Calcium ions are shown at the top and bottom of both domains they interact with. Images made using the program SETOR (34). Supporting Figure 2. (A) Ribbon diagrams for domain pairs, EC1-EC2, EC2-EC3, EC3-EC4 and EC4-EC5. The top domains are presented in the same orientation to demonstrate the relative twist between domain pairs. EC3-EC4 demonstrates a substantially different twist than the other three domain pairs. β-strands are colored blue and pink to notate the front and back β-sheets of each domain (front face includes strands A*, A, C, F and G; back face includes strands B, D and E). The green ribbon represents the quasi-β-helix. The yellow ribbon represents the α-helix in domain EC2. The side chain of Trp 2 is colored in olive green, calcium ions are green and disulfides are yellow. (B) Interdomain rotations are illustrated through a ribbon diagram representation of the whole molecule in the same orientation as Fig. 1A. The green ribbon in EC1 highlighted by a green arrow depicts the quasi-β-helix region. The yellow ribbon shows the only α- helical region, at the top of domain EC2. Trp 2 is drawn in stick representation and colored in olive green; calcium ions are green, and disulfides are cyan. Purple density indicated by the purple arrow depicts 4σ Bijvoet difference Fourier density for the 6Å resolution ytterbium data set. Images made using the program SETOR (34). Boggon et al. Page 10

11 Supporting Figure 3. The four calcium binding sites. High-resolution structural studies have shown that the sequence repeats of cadherin extracellular segments fold into domains with an immunoglobulin-like topology (9, 14, 15). The connections between these domains are rigidified by the ligation of three Ca 2+ ions by conserved residues at the bottom of domain n, the top of domain n+1, and the linker segment between them (10, 16, 17). All side-chains directly involved in calcium binding sites are shown and labeled (carbonyls are not shown). Calcium ions, shown as green spheres, are labeled according to convention (10). A conserved proline at the start of each domain defines the base of each calcium binding site. Gln 365 is colored purple, as this calcium binding location is occupied by a carbonyl in the other three binding sites. Images made using the program SETOR (34). Supporting Figure 4. Sequence alignments for Classical/type I cadherins (C-, E-, P-, N-, R-, M-, T-cadherins); Desmocollins 1, 2, and 3; Desmogleins 1, 2 and 3; and a selection of type II cadherins (OB- cadherin, K-cadherin, and cadherin-8). All sequences are human apart from C-cadherin, which is from Xenopus Laevis. Regions of high sequence identity (at least three matches) are shaded yellow. The observed secondary structure is shown and labeled with front β-strands colored red and back β-strands colored blue. A small β-strand conserved in domains EC2 through EC5 is labeled A*. The quasi-β-helix in EC1 is colored green and the α-helix in EC2 is colored yellow. Calcium binding residues are boxed in green and calcium binding carbonyls have a green triangle above. Glycosylated residues are indicated by a brown oval above. Disulfide bonds are boxed in cyan and the appropriate linkages are indicated. A conserved Pro at the start of each domain is boxed in dark purple. Residues involved in the strand dimer interaction are indicated, with residues from the Trp 2 donor side having a green box above and from Boggon et al. Page 11

12 the Trp 2 acceptor side a blue box. Residues involved in the EC1-EC2 cis interaction are indicated with a red box above. Point mutations reported in the literature are boxed for the cadherin in which they were observed. A red box indicates the mutation abolished adhesion, a blue box indicates the mutation does not abolish adhesion, a light purple box indicates the mutation is seen in tumors. The mutations are listed and color-coded in the table of mutations, and are taken from the literature (5, 16, 17, 21, S7). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. GenBank database accession codes are as follows: C-cadherin, P33148; E-cadherin, P12830; P-cadherin, P22223; N- cadherin, P19022; R-cadherin, P55283; M-cadherin, P55291;T-cadherin, P55290; Desmocollin-1, Q08554; Desmocollin-2, Q02487; Desmocollin-3, Q14574; Desmoglein- 1, Q02413; Desmoglein-2, Q14126; Desmoglein-3, P32926; OB-cadherin, P55287; K- cadherin, P55285; Cadherin-8, P Sequence alignment made with the program DNASTAR (S8). Supporting Figure 5. The strand dimer interface. (A, B, C and D) show EC1-EC1 interactions in C-cadherin (this work), N-cadherin (pdb code: 1NCG), N-cadherin (pdb code: 1NCH-B) and N-cadherin (pdb code: 1NCI) respectively. The strand dimer is observed in a number of unrelated space-groups. Domain orientation angles show the twist between the two opposing domains (obtained using the CCP4 program HELIXANG (36)). Trp 2 side-chains are shown and calcium ions are green spheres. (E) Overlays of the strand dimer observed in C-cadherin and N-cadherin (1NCH-A), with one domain of the dimer superposed, illustrating the flexibility of the strand dimer. C-cadherin is Boggon et al. Page 12

13 colored in pink and red, N-cadherin in blue and cyan. Images made using the program SETOR (34). Supporting Figure 6. (A) Backbone worm trace of two C-cadherin ectodomains connected through a strand dimer interaction (yellow), and the orientation of C-cadherin ectodomains (brown) when their EC1 domains are superposed on an N-cadherin strand dimer (1NCH-A). Both structures depict interactions in conformations that are largely trans. Even the more acute angles defined by the N-cadherin strand dimer define a dimer pair that is essentially antiparallel, and appears incompatible with partner molecules being anchored in the same membrane. This suggests that formation of strand dimers between cadherins emanating from the same cell surface would require either substantial flexibility of the calcium-rigidified ectodomain, or a greater range of plasticity in the strand dimer interface than has been seen in the N- and C-cadherin structures. Calcium ions are shown as green spheres, and disulfide bonds are drawn in purple. (B) View from 90 away. Images made using the program SETOR (34). Boggon et al. Page 13

14 Supporting Tables Supporting Table 1. Root-mean-square distances (RMSD) (Å) between equivalent alpha carbon atoms generated within a distance of 5Å of one another chosen automatically within the program LSQMAN (S9). The number of equivalent alpha carbon atoms is shown in parentheses. Domains EC1 and EC5 show the greatest structural diversity, with fewer equivalent alpha carbon atoms and higher RMSDs than superpositions between EC2, EC3 and EC4. Domain EC2 EC3 EC4 EC5 EC (75) 1.90 (72) 1.62 (77) 2.05 (48) EC (93) 1.53 (90) 1.76 (64) EC (83) 1.91 (72) EC (77) Boggon et al. Page 14

15 Supporting Table 2. Schematic representation of angular relationship between domains. Angles Tilt, Skew and Twist define the degrees of freedom of the rotational domain, using its N-terminus as a pivot point, when compared to the reference domain. The cadherin domains and their residue limits are indicated in the columns Domain and Residues. A rigid frame was calculated for each individual domain with the center of mass as the origin. The z-axis of each domain was aligned with the main axis of the inertia tensor calculated using backbone atoms and pointed towards the C-terminus end of that domain. The x-axis was defined as pointing from the center of mass towards an alpha carbon of a residue in the middle of β-strand G, this residue was equivalent in all domains and is indicated in columns Residue defining x. Angles are defined as follows. Tilt: tilt required to align the z-axis of the rotational domain with that of the reference domain. Skew: direction with respect to the x-axis of the reference domain that the tilt is made in. Twist: angle required to align the x-axis of the rotational domain with that of the reference domain. Angles Tilt, Skew and Twist were calculated using a program written by Bruno Kieffer (Strasbourg, France) (S10, S11). The relationship between EC1 and EC2 for all structures seems to be approximately equivalent, with tilt ~20, skew~-40 and twist ~-120. In C-cadherin the relationship between EC2 and EC3 is again approximately equivalent to that between EC1 and EC2, with a somewhat divergent skew. The relationship between EC3 and EC4 displays a significantly sharper tilt between the z-axes skewed in an almost opposite direction to the interactions between EC1/EC2 and EC2/EC3. The relationship between EC4 and EC5 displays a slightly less sharp tilt than that of EC3/EC4. Twist angles are especially conserved between all consecutive domains, showing a rotation of approximately a third ( ) between the consecutive x-axes. There is approximately a 100 tilt between the z-axes of domain Boggon et al. Page 15

16 EC1 and domain EC5 of C-cadherin. This analysis highlights the observation that EC3/EC4 interaction is somewhat divergent from the other domain pair interactions. Boggon et al. Page 16

17 C-Cadherin (this work) E-Cadherin 1EDH E-Cadherin 1FF5 Reference Domain Rotational Domain TILT SKEW TWIST Residue Residue Domain Residues Domain Residues ( ) ( ) ( ) defining x defining x EC EC EC EC EC EC EC EC EC EC EC1 - A EC2 - A EC1 - B EC2 - B EC1 - A EC2 - A EC1 - B EC2 - B N-Cadherin 1NCJ EC EC Boggon et al. Page 17

18 Supporting Table 3. Inter-molecular interactions observed in the strand dimer interface. Backbone atoms are indicated by BB, and side chain atoms by SC. Distances are in angstroms. -- indicates that one or both atoms is not seen in the electron density. Distances calculated in O (S4). Interacting Atoms Crystal Structure Atom 1 Atom 2 CCAD 1NCG 1NCH-A 1NCH-B 1NCI-A 1NCI-B N -ter (BB) OE2 Glu 89 (SC) N -ter (BB) OD1 Asn 27 (SC) O Asp 1 (BB) N Asn 27 (BB) OD2 Asp 1 (SC) NE Arg 28 (SC) NE1 Trp 2 (SC) O Gln 90 (BB) N Val 3 (BB) O Lys 25 (BB) O Val 3 (BB) N Lys 25 (BB) NZ Lys 8 (SC) OE1 Gln 23 (SC) O Ser 57 (BB) NE Arg 23 (SC) NH2 Arg 25 (SC) O Arg 23 (BB) NH2 Arg 28 (SC) OE2 Glu 89 (SC) NH1 Arg 28 (SC) O Val 88 (SC) OD1 Asp 29 (SC) NH1 Arg 25 (SC) O Gly -2 (BB) NH2 Arg 28 (SC) Residue 23 is Arg in N-cadherin and Gln in C-cadherin Residue 27 is Gly in N-cadherin and Asn in C-cadherin Residue 28 is Arg in N-cadherin and Lys in C-cadherin Residue 57 is Ser in N-cadherin and Thr in C-cadherin Residue 90 is Asn in N-cadherin and Gln in C-cadherin Boggon et al. Page 18

19 Supporting Table 4. Ramachandran angles, in degrees, of residues in the A-strand involved in strand dimer formation. Angles χ 1 (rotation around the C α C β bond) and χ 2 (rotation around the C β C γ bond) are also shown for Trp 2. Angles are shown for all molecules in each high-resolution cadherin structure solved to date, with molecules A and B corresponding to those in the PDB depositions. N-cadherin structures 1NCG, 1NCH and 1NCI are domain EC1 only. E-cadherin structures 1EDH and 1FF5 contain domains EC1 and EC2. Ramachandran angles were calculated in O (S4). Where values are not given for Trp 2 and Val 3, there are no coordinates for the relevant residues in the corresponding PDB file. For those structures with constructs not starting at Asp 1 (i.e. with an extraneous Met or Gly-Ser at the N-terminus), 1NCH-A, 1NCI-A and 1NCI-B all displayed Gly 1, Ser 2 in stable density, and 1FF5-A and 1FF5-B both reported Met 1 in density. The Trp 2 side chain was not visible in the 1EDH-A and 1EDH-B structures. Trp 2 is inserted into the hydrophobic pocket of its own EC1 domain in structures 1FF5- A and 1FF5-B. Neither the 3.4 Å structure 1NCJ nor the NMR-derived structure 1SUH are of sufficient quality to yield reliable Ramachandran angle information, so they have been omitted from this table. Trp 2 Val 3 Ile 4 Pro 5 Pro 6 Ile 7 φ ψ χ 1 χ 2 φ ψ φ ψ φ ψ φ ψ φ ψ CCAD NCG NCH-A B NCI-A B EDH-A B FF5-A B Boggon et al. Page 19

20 Boggon et al. Page 20

21 Supporting Table 5. Occurrences of strand and cis dimers in all cadherin crystal structures. Number of domains crystallized is shown. The angle between strand dimerconnected domains is also given, as well as the distance between C-terminus proximal alpha carbon residues 99 and its strand dimer mate 99 from the partner molecule. Some modifications at the N-terminus arising from cloning artifacts are noted. Buried surface areas were calculated using the program Grasp (35). Crystal Structure Number of Domains C-cadherin (this work) 5 Observed Angle = 88 Distance = 33Å Strand Dimer Observations Buried surface = 2230Å 2 N-cadherin 1NCG 1 Observed Angle = 61 Distance = 21Å Buried surface = 1880Å 2 N-cadherin 1NCH 1 Observed Angles = 53 and 63 Distances = 20Å and 23Å Buried surfaces = 2400Å 2 and 1870Å 2 N-cadherin 1NCI 1 Observed Angle = 54 Distance = 22Å Buried surface = 2280Å 2 Cis Dimer Observations Observed Buried surface = 1680Å 2 Not-observed (Only one domain) Not-observed (Only one domain) Not-observed (Only one domain) N-cadherin 1NCJ 2 Not-observed Not-observed E-cadherin 1EDH 2 Not-observed (N-terminus of construct contained additional residues Met-Arg) E-cadherin 1FF5 2 Not-observed (N-terminus of construct contained additional residue Met) Observed Buried surfaces = 1320Å 2 and 1380Å 2 Observed Buried surface = 1240Å 2 Boggon et al. Page 21

22 Supporting References S1. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307 (1997). S2. A. T. Brünger, X-Plor Version 3.1 Manual (Yale University, New Haven, 1993). S3. A. T. Brünger, et al., Acta Crystallogr. D 54, 905 (1998). S4. T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta. Crystallogr. A 47, 110 (1991). S5. Y. Luo, et al., Development 128, 459 (2001). S6. S. C. Suzuki, T. Inoue, Y. Kimura, T. Tanaka, M. Takeichi, Mol. Cell Neurosci. 9, 433 (1997). S7. M. Ozawa, J. Engel, R. Kemler, Cell 63, 1033 (1990). S8. DNASTAR, Mol. Biotechnol. 5, 185 (1996). S9. G. J. Kleywegt, T. A. Jones, in From First Map to Final Model S. Bailey, R. Hubbard, D. Waller, Eds. (SERC Daresbury, Warrington, 1994) pp S10. A. P. Wiles, et al., J. Mol. Biol. 272, 253 (1997). S11. P. Bork, A. K. Downing, B. Kieffer, I. D. Campbell, Q. Rev. Biophys. 29, 119 (1996). Boggon et al. Page 22