Insights into the Low Adhesive Capacity of Human T-cadherin from the NMR Structure of Its N-terminal Extracellular Domain*

T-cadherin is unique among the family of type I cadherins, because it lacks transmembrane and cytosolic domains, and attaches to the membrane via a glycophosphoinositol anchor. The N-terminal cadherin repeat of T-cadherin (Tcad1) is ≈30% identical to E-, N-, and other classical cadherins. However, it lacks many amino acids crucial for their adhesive function of classical cadherins. Among others, Trp-2, which is the key residue forming the canonical strand-exchange dimer, is replaced by an isoleucine. Here, we report the NMR structure of the first cadherin repeat of T-cadherin (Tcad1). Tcad1, as other cadherin domains, adopts a β-barrel structure with a Greek key folding topology. However, Tcad1 is monomeric in the absence and presence of calcium. Accordingly, lle-2 binds into a hydrophobic pocket on the same protomer and participates in an N-terminal β-sheet. Specific amino acid replacements compared to classical cadherins reduce the size of the binding pocket for residue 2 and alter the backbone conformation and flexibility around residues 5 and 15 as well as many electrostatic interactions. These modifications apparently stabilize the monomeric form and make it less susceptible to a conformational switch upon calcium binding. The absence of a tendency for homoassociation observed by NMR is consistent with electron microscopy and solid-phase binding data of the full T-cadherin ectodomain (Tcad1-5). The apparent low adhesiveness of T-cadherin suggests that it is likely to be involved in reversible and dynamic cellular adhesion-deadhesion processes, which are consistent with its role in cell growth and migration.

homologous cadherin on an opposing cell. The calcium-dependent adhesion mechanism was first identified in N-and E-cadherin (21,23) and recently confirmed for other cadherins (19,24,25). The alignment of amino acid sequences of the N-terminal extracellular domain of human T-and classical cadherins reveals Ϸ30% sequence identity (Fig. 1B). Residues important for calcium binding such as Glu-11 and the calcium binding motifs LDRE and DXND are well conserved. In contrast, amino acids important for the adhesive function of classical cadherins are not shared by T-cadherin (Fig. 1B). These include Trp-2 and residues of the hydrophobic binding pocket of cad1 (Ile-24, Ser-26, Tyr-36, Ala-80, Asp-90, and Met-92), which accommodates Trp-2 in the intercellular strand-exchange dimer (19 -21, 26, 27). Similarly, residues in cad1 and cad2 that make contacts in the crystal structure of C-cadherin (19) and which have been proposed to represent the cis-interaction interface are mostly replaced by other amino acids ( Fig.  1B and supplemental Fig. S1). Based on the unusual amino acid composition, domain organization, and different cellular localization, it has been suggested that the adhesive capacity of T-cadherin is lower and that the mechanism of T-cadherininduced calcium-dependent cell-cell adhesion should be different from those of classical cadherins (28).
Here we report the NMR characterization of the structure, dynamics, and calcium binding properties of the N-terminal cadherin domain of human T-cadherin (Tcad1, residues 1-105 of the mature form, residues 139 -243 of the unprocessed precursor protein). Tcad1, as the other characterized cadherin domains, adopts a ␤-sandwich structure with a Greek key folding topology. However, Tcad1 is monomeric, both in the absence and presence of calcium. This is supported by additional EM and solid-phase binding data of the full T-cadherin ectodomain . The structure of Tcad1 shows that isoleucine 2, which is conserved in T-cadherins from different organisms (Fig. 1C), occupies a hydrophobic pocket on the same monomer. The latter is formed by equivalent residues to those lining the binding pocket for Trp-2 in the strand-exchange dimer of classical cadherins. Based on the presented NMR data of Tcad1, the higher stability of the monomeric form and its lower susceptibility for calcium-induced conformational changes are rationalized.

EXPERIMENTAL PROCEDURES
DNA Constructs for NMR Studies-The DNA fragment encoding for the first two extracellular domains of T-cadherin (1-223, Tcad12) preceded by a His tag/Factor Xa cleavage site (MGH 10 SSGHID 4 KHMIEGR) was obtained by PCR using a cDNA of human T-cadherin. The fragment was cloned into NdeI/BamHI restriction sites of the Escherichia coli expression vector pET-19b (Novagen). A plasmid encoding residues 1-105 (Tcad1) was prepared by introducing a stop codon after residue 105 in the Tcad12-encoding plasmid.
Expression and Purification of Tcad1 and Tcad12 for NMR Studies-Proteins were expressed in E. coli BL21 (DE3) cells (Novagen). 15 N and 13 C labeling was carried out by growth on minimal medium using 15 NH 4 Cl, and 13 C 6 -glucose as the sole nitrogen and carbon sources, respectively. Cells were disrupted in 50 ml of 50 mM Tris, 50 mM NaCl, pH 8 using a French press. The resulting protein solution was diluted 1:1 and the protein denatured by the addition of 6 M urea. The unfolded protein was loaded onto a Ni-affinity chromatography column. Unspecifically bound proteins were removed by washing the column with 50 mM Tris, 50 mM NaCl, 6 M urea, pH 8. Refolding of the protein on the column was achieved by washing with the same buffer without urea. Elution occurred in two steps by raising the imidazole concentration (300 and 500 mM). Tcad1 was digested without dialysis after 1:1 dilution with an imidazole-free buffer. Tcad12 was dialyzed into 20 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 6.8 prior to digestion with Factor Xa. In both cases, the cleaved His tag was removed by Ni-affinity chromatography. The correct mass was confirmed by MALDI-TOF or ESI mass spectrometry.
Structure Calculation-All structure calculations were performed with XPLOR-NIH (33) using torsion angle molecular dynamics. Distance restraints were generated in NMRView and classified according to NOE-cross-peak intensities. Upper bounds were 2.8, 3.5, 4.5, and 5.5 Å. The lower bound was always 1.8 Å. For all NOE-restraints r Ϫ6 sum averaging was used. Backbone dihedral angle restraints for and were derived based on 3 J HNH␣ , the determined 13 C and 1 H ␣ chemical shifts, and on initial structure calculations. Stereospecific assignments were obtained for 13 ␤-methylene and 8 valine ␥-methyl proton pairs. Based on 3 J H␣H␤2/3 and 3 J N␤2/3 coupling constants and NOE data, side chain 1 angles were restrained to one of the staggered conformations (60°, 180°, Ϫ60°) Ϯ 30°. For regions with ␤-sheet conformation, hydrogen bonds (H-bonds) were defined by HN-O distance bounds of 1.8 -2.3 Å, and N-O distance bounds of 2.6 -3.1 Å. The 20 lowest energy structures out of 200 calculated ones were finally refined in a water shell (34,35).
Protein Expression and Purification for EM and Solid-phase Binding Studies-Several (fusion) protein constructs were generated to carry out in vitro EM and solid-phase binding studies (Fig. 1A). These comprise His-tagged (-H) and Strep-tagged (-S) versions of T-cadherin domains 1-3 (Tcad1-3-S), fusions containing the first five domains of T-cadherin and the coiledcoil trimerization domain of cartilage matrix protein (Tcad1-5cmp-H/S), a fusion containing the first T-cadherin domain, domains 2-5 of E-cadherin, and the trimerization domain of cartilage matrix protein (Tcad1Ecad2-5cmp-H), as well as a fusion containing the first five domains of E-cadherin and the trimerization domain of cartilage matrix protein (Ecad1-5cmp-H) or the pentamerization domain of cartilage oligomeric matrix protein (Ecad1-5comp-S). Vectors for expression of these constructs in human embryonal kidney cells were obtained as described in the supplemental data.
Human embryonal kidney (HEK) 293-EBNA cells (Invitrogen) were cultured in Dulbecco's modified Eagle's medium (DMEM) F12 supplemented with 10% fetal bovine serum, 1% glutamine and 10 mg/ml penicillin/streptomycin (all obtained from Invitrogen). Transfectants were generated as described before (36), and bulk cultures were used for protein expression. Confluent cells were maintained in 23 ml of DMEM F12, 1% glutamine, 10 mg/ml penicillin/streptomycin for 48 h. Then the medium was collected, and new medium was added. This procedure was repeated 8 -10 times. Supernatants were centrifuged at 2500 ϫ g for 10 min, buffered in 20 mM Hepes, pH 7.1, and stored at Ϫ20°C.
Solid-phase Binding Assays-200 l of trimeric Ecad1-5cmp-H (50 nM), Tcad1-5cmp-H, or Tcad1Ecad2-5cmp-H (50 nM) in 20 mM Tris, pH 7.4 were coated at 4°C overnight on 96-well flat bottom plates (Becton Dickinson). All buffers contained 2 mM Ca 2ϩ . Plates were then blocked with 20 mM Tris, pH 8.1 and 3% (w/v) bovine serum albumin for 2 h at room temperature. After three washing steps, different concentrations of the Strep-tagged ligands Ecad1-5comp-S, Tcad1-3-S, or Tcad1-5cmp-S (in 20 mM Tris, 0.04% Tween 20, 2 mM Ca 2ϩ ) were added to the wells for 2 h at room temperature. Wells were again washed three times before 1 mg/ml HRP-conjugated streptavidin was added for 1.5 h at room temperature. HRP activity was measured at 405 nm using ABTS (Roche Applied Science) as substrate. One representative experiment out of three (almost identical) is shown.

RESULTS
Description of the Structure of Tcad1-Initial NMR studies on a T-cadherin construct containing the first two cadherin repeats (Tcad12) indicated that the second repeat was unfolded (supplemental Fig. S2). The reason for this behavior is currently unclear. Under similar conditions, the second domain of Ecad12 with equivalent domain boundaries (1-219) is folded (22). Therefore the domain boundaries should have been defined correctly. It has however been observed that in an E-cadherin construct encompassing the 2nd and the 3rd cadherin repeat, the latter is unfolded (37). Because the second domain in Tcad12 is unfolded, we restricted the structural characterization to Tcad1. According to NMR relaxation, EM, and solid-phase binding data, this Tcad1 domain is monomeric in solution in the presence and absence of calcium (see below).
In comparison to classical cadherins, the most interesting part of the Tcad1 structure is the region around isoleucine 2. In Tcad1, residues Ile-2 and Val-3 (strand A, Fig. 2A) form an  ). B, alignment of the amino acid sequences of the N-terminal extracellular domain from different type I cadherins (t, T/truncated/heart; e, E/epithelial; n, N/neuronal; r, R/retinal; c, C/EP) from human (h) and frog (xl). Residues involved in calcium binding are marked by green bars below the sequence alignment. Trp-2 (W, red bar) that is conserved in classical cadherins is important for the formation of the strand-exchange dimer during cell-cell adhesion. In E-cadherin, the respective binding pocket is formed by residues Ile-24, Ser-26, Tyr-36, Ala-80, Asp-90, and Met-92 (blue bars). In classical cadherins Glu-89 (pink bar, Glu-88 in tcad) can form an intermolecular salt-bridge with the N-terminal amide group. The structure of C-cadherin revealed an intermolecular hydrogen bond between the side chains of Lys-8 and Gln-23 (orange bars). Based on the crystal structure of C-cadherin, the cis contact interface between cad1 and cad2 involves the sequence positions labeled by black bars. C, N-terminal cadherin repeat of T-cadherin (Tcad1) is conserved to Ϸ70% between species (h, human; o, orangutan; b, bovine; c, chicken; m, mouse; r, rat). Sequence alignment representations were generated using the program ESPript (48).
antiparallel ␤-sheet with residues Val-25 and Asp-26 (strand D). This is evident from interstrand NOEs between V2H ␣ and V26H ␣ , V3H N and V25H N , and strong sequential H ␣ i-1 to H N i contacts. Accordingly, in all 20 structures, V3N and V25O as well as V25N and V3O are within H-bonding distance to each other. In Ecad1, NMR evidence shows that V3N and the equivalent K25O also form an intramolecular H-bond for monomeric E-cadherin at low concentrations (22). Such an intramolecular H-bond is also observed in the ECAD1-internalin complex (40). At high concentrations in the presence of calcium, classical cadherins dimerize via strand-exchange, and the V3N-K25O H-bond switches to an intermolecular form. The resulting dimeric H-bonds are basically identical in all structures that show strand-exchange, i.e. N-cadherin (21), C-cadherin (Fig. 3B, right side) (19), and E-cadherin (22). For T-cadherin, no evidence of dimerization is observed, but apparently the monomeric T-cadherin preserves the H-bond contact between Val-3 and residue 25 seen in monomeric forms of other cadherins.
The side chain of Ile-2 in Tcad1 inserts into a pocket that is formed by residues equivalent to those lining the hydrophobic pocket for Trp-2 in classical cadherins (Fig. 3,  B-D). Mostly hydrophobic interactions are found to residues Val-24, Asp-26, Arg-29, Phe-35, Val-77, Thr-79, Glu-88, and Val-91. Additional contacts involve the backbone of Val-25, Ser-27, Glu-78, Gly-89, and Pro-90 (Fig. 3, B, left side and  C). In the structures of the C-, and E-cadherin, the conserved side chain indole and hydroxyl groups of Trp-2 and Tyr-36 donate hydrogen bonds to the backbone carbonyl groups of Asp/Glu-90 and Ser-26, respectively (Fig. 3B, right). These hydrogen bonds cannot be formed by the equivalent Ile-2 and Phe-35 in Tcad1 due to the absence of a hydrogen bond donor (Fig. 3B, left side). The structure of C-cadherin (Fig. 3, A and B, right side) revealed a further hydrogen bond between the side chains of Lys-8 and Gln-23 (19). Also, this hydrogen bond cannot be realized in Tcad1 because position 8 is occupied by a leucine.
It has been shown that strand-exchange during cadherin dimer formation depends on the formation of an intermolecular salt bridge between the N-terminal amino group and the side chain of Glu-89 (41). An equivalent glutamic acid is found at position 88 in T-cadherin. In Tcad1, the N-terminal nitrogen and the side chain oxygen atoms of Glu-88 may form an intramolecular salt bridge, because their distance is Յ 2.6 Å in 6 of the 20 final structures. However, the N terminus may also be involved in other ionic interactions (Fig. 3B, left side), i.e. the distance to the side chain oxygen atoms of the strongly conserved Asp-26 (Fig. 1C) is Յ2.5 Å in all calculated structures and the distance to side chain oxygen atoms of the fully conserved Asp-28 is Յ 2.5 Å in 8 of 20 structures. These additional ionic interactions may thus stabilize the monomeric form and counteract the intermolecular interaction with Glu-88, which facilitates dimer formation in classical cadherins. Moreover, T-cadherin has a serine instead of an aspartate at position 1 (Fig. 1B). In classical cadherins this conserved negatively charged residue may further influence the monomer-dimer equilibrium, e.g. by repulsive intramolecular interactions with Glu-89.
It is revealing to compare the surface charge of monomeric Tcad1 (Fig. 3C) to other monomeric cadherin structures. Fig.  3D shows the surface charge of cad1 in monomeric M-Ecad12,  AUGUST 22, 2008 • VOLUME 283 • NUMBER 34

JOURNAL OF BIOLOGICAL CHEMISTRY 23489
which has an N-terminal extra methionine that abolishes its ability to form the strand-exchange dimer (22). In this structure, Trp-2 is inserted into the Trp-2 binding pocket on the same molecule. Hence, the surface of this molecule should closely mimic the surface of monomeric Ecad12 before it associates into the strand-exchange dimer. It is apparent from this electrostatic surface that strand-exchange is strongly favored by a high density of opposite charges across the entire contact surface around residue Trp-2. In contrast, the surface around Ile-2 in T-cadherin is more hydrophobic toward the bottom part of the molecule (C terminus) and rather acidic toward the top part. Thus, no favorable electrostatic interactions appear possible for the formation of a strand-exchange dimer.
Chemical Shift Changes upon Calcium Binding-To characterize the effect of calcium binding on the structure and the adhesive properties of Tcad1, we analyzed 15 N-HSQC spectra of 0.2 mM Tcad1 at different calcium concentrations (supplemental Fig. S4). At 0.5 mM Ca 2ϩ , only weak shifts are observed, which are most pronounced for residues 67-70 in one of the calcium binding motifs. At Ca 2ϩ concentrations of 5-10 mM, the equilibrium is completely driven to the calcium-bound form. Resonances of residues at the calcium binding sites (Glu-11-Asn-12, Leu-65-Ile-70, Ile-98 -Asn-101) shift strongly (Fig. 4). Further, nearby residues show only weak to very weak displacements. No significant changes are observed for the N-terminal region around residue Ile-2 that would indicate structural rearrangements.
Calcium binding to E-cadherin significantly increases its affinity for homoassociation and its adhesiveness (23,42). For Ecad12, homoassociation leads to distinct changes in NMR spectra (22), such that monomeric and dimeric forms can be clearly distinguished. Both calcium-free and -bound forms undergo dimerization. However, the respective dissociation constant (K D ) is much stronger (0.72 mM) in the presence of calcium than in its absence (10 mM) (22).
Addition of calcium to Tcad1 (0.2 mM) did not result in a decrease of the amide proton T 2 relaxation times (ϳ18.5-22.5 ms), but made them overall more uniform (ϳ22.5 ms). These data are consistent with a monomeric form of calcium-free and -bound Tcad1 and suggest that calcium binding results in compaction of the molecule due to reduced conformational freedom at the calcium binding sites. Moreover, a variation of the  concentration of calcium-free Tcad1 (0.07-0.6 mM) did not induce any changes in the 1 H-15 N HSQC spectra. This is in contrast to Ecad12 where such changes are clearly detectable even in the absence of calcium (22). 15 N-relaxation data (supplemental Fig. S2) obtained on calcium-free Tcad12 (24.7 kDa) at a concentration of 0.86 mM yield an overall rotational correlation time of 11.7 ns. The latter is also consistent with a monomeric form. Thus the NMR evidence indicates that both Tcad12 and Tcad1 are monomeric in the absence of calcium up to at least millimolar protein concentrations. Because the addition of calcium at these protein concentrations does not give any evidence for homoassociation, we conclude that any putative association either in the presence or absence of calcium should be much weaker than for the case of calciumfree Ecad12 (K D , 10 mM).
EM and Solid-phase Binding Data for Tcad1-5 and Ecad1-5-As the NMR data on Tcad1 and Tcad12 gave no evidence for dimerization, we additionally analyzed homophilic interactions of T-cadherin constructs containing the full ectodomain (cad1-5, Fig. 1A) by EM and solid-phase binding assays. To increase the local cadherin concentration and thereby to mimic the conditions on the cell surface, most of the used E-and T-cadherin constructs were fused at their C terminus to the coiled-coil trimerization domain of cartilage matrix domain (cmp in the construct name in Fig. 1A; a schematic representation of such a construct is depicted in supplemental Fig. S5A).
Calcium binding to classical cadherins results in characteristic changes in their appearance in electron micrographs. Binding of calcium at low concentrations (0.5-1 mM) stiffens the linkers between the five cadherin repeats and induces a characteristic curvature of the entire extracellular structure (23). The latter is also evident from the crystal structure of C-cadherin (Fig. 3A) (19). At higher calcium and protein concentrations, the electron micrographs of E-cadherin show ring-like and concatenated ring structures when ectodomains are tethered together by oligomerization domains (23). An analogous Tcad1-5cmp construct did not show the formation of such concatened ring-like structures in electron micrographs under similar conditions (supplemental Fig. S5B). This further corroborates that T-cadherin has a much lower tendency for homodimerization than classical cadherins.
The adhesive capacity of different E-and T-cadherin constructs in the presence of calcium was further analyzed by solidphase binding assays (Fig. 5). In these assays, various cadherin constructs (Ecad1-5cmp-H, Tcad1-5cmp-H, Tcad1Ecad2-5cmp-H, see Fig. 1A) were coated to the bottom of the reaction vial. These were reacted with Strep-tagged counterparts (Ecad1-5comp-S, Tcad1-3-S, Tcad1-5cmp-S) added in solution. Subsequent binding of streptavidin-attached HRP to the Strep tag could be monitored by UV-spectroscopy based on the catalytic activity of HRP. The addition of Ecad1-5comp-S to coated Ecad1-5cmp-H (Fig. 5A, open circles) resulted in a strong hyperbolic increase of the absorption of the ABTS substrate at 405 nm, which indicates strong homoassociation. Because of the specificity of cadherin interactions, no significant binding of Ecad1-5comp-S to coated Tcad1-5cmp-H (Fig. 5A, black circles) or the domain exchange mutant Tcad1Ecad2-5cmp was observed (Fig. 5A, open triangles). When immobilized Tcad1-5cmp-H, Ecad1-5cmp-H, or Tcad1Ecad2-5cmp-H was incubated with increasing concentrations of monomeric Tcad1-3-S (up to 400 nM, Fig. 5B); also no binding was detected. Neither did Tcad1-5cmp-S react with coated Tcad1-5cmp-H, the chimera Tcad1Ecad2-5cmp-H, nor Ecad1-5cmp-H (Fig. 5C). Taken together, the solid-phase binding data give no evidence of homoassociation of trimerized immobilized T-cadherin ectodomains to soluble T-cadherin in the submicromolar range. This is consistent with the absence of any NMR-detectable interactions of the Tcad1. Binding was monitored at 405 nm after incubation with 1 g/ml HRP-conjugated streptavidin and ABTS as substrate. One typical experiment is depicted. Data points represent the mean value Ϯ S.D. of four wells corrected by the negative control (unspecific binding to bovine serum albumin alone).

DISCUSSION
The N-terminal extracellular domain of classical type I cadherins mediates cell-cell adhesion by formation of a strand-swapped intercellular dimer. Trp-2 from one monomer binds thereby into a hydrophobic pocket presented by the equivalent domain of a cadherin molecule on a neighboring cell. The presented structural data of Tcad1 reveal how several features conserved in the amino acid sequences of T-cadherins from different organisms but distinct to those of other type I cadherins (Fig. 1) reduce its ability for dimerization. First, Tcad1 has an isoleucine instead of a tryptophan at position 2 and several of the residues forming the hydrophobic pocket for residue 2 have larger side chains  (44). The ␤-sheet like -angle of Ser-5 in Tcad1 may therefore exert less conformational strain on the preceding N-terminal ␤-strand than Pro-5 in classical cadherins. Third, Tcad1 has a glutamine instead of a glycine at position 15. It has been described that the formation of the strand-exchange dimer in classical cadherins has the largest effect on the backbone conformation of residues 14 -16 (20). Moreover the -angles of residues 17-18 differ significantly between the closed monomer of M-E-cad (PDB ID 1FF5) and the dimer of mature E-cad12 (PDB ID 1Q1P). In the known structures of classical cadherins, Gly-15 has positive or largely negative -angles. Such -angles are typically not observed for glutamine. In Tcad1, the reduced backbone flexibility around Gln-15 (Fig. 3C, left and supplemental Fig. S2) is expected to hamper strandexchange upon dimer formation by making T-cadherin less susceptible to conformational changes upon binding of calcium to the spatially close binding sites (Figs. 3C and 4). Fourth, studies with N-cadherin highlighted the relevance of an intermolecular salt-bridge between the N-terminal positively charged amide group and the negatively charged carboxylate of Glu-89 for dimer formation (41). Based on the observed distances in the calculated Tcad1 structures, ionic interactions between the N-terminal amide and Asp-26 as well as Asp-28 occur more often than with Glu-88 (Fig. 3B,  left side). This may additionally stabilize the formation of a stable ␤-sheet around Ile-2 and lower the affinity for homoassociation. Fifth, the exchanges of Asp to Ser at position 1 and Lys/Arg to Val at position 25 change the surface charge distribution in the vicinity of residue 2. Whereas dimer formation in E-cadherin may be driven by favorable ionic interactions involving complementary surface patches (Fig. 3D, middle), such complementary charges are absent in T-cadherin (Fig. 3C, middle).
T-cadherin does not only lack many of the sequence features of classical cadherins, which are important for the trans interaction between the two cad1 repeats from opposing cells. T-cadherin also exhibits significant differences in the surface patch distal of position 2, which is thought to mediate the so-called cis contact with a cad2 repeat of a neighboring molecule on the same cell (Fig. 3A, left side). Overall, this surface region of Tcad1 (Fig. 3C, black circled region) has a similar shape but a different polarity compared with E-and C-cadherin. In classical cadherins, the first and the last residue of the HAV-motif (79 -81) as well as Ser-37, X-55, and Asn/Ser-86 of cad1 (Fig. 1B) mediate interactions to residues Ile/Val-174 and Ala/Thr-176 on cad2 (supplemental Fig. S1). In Tcad1, the equivalent surface on cad1 is formed by an ETT motif (78 -80) as well as Arg-36, Glu-54, and Lys-85. The surface on the second domain is formed by Ser-175 and Ala-177. However, two two-residue insertions (Thr-172-Val-173, Thr-183-Leu-184) and the substitution G178L are expected to significantly alter the surface characteristics of Tcad2 in this region. The increased polarity on cad1 and the presumably different shape of the surface on cad2 might therefore result in reduced complementarity and thereby weaker cis interactions.
In the current model of cadherin-mediated cell adhesion, the closed monomer, in which residue 2 binds into the hydrophobic pocket of the same protomer, competes with the strand-exchange dimer, where residue 2 binds into the hydrophobic pocket of a protomer on an opposing cell. Because the interactions in the monomeric and dimeric forms are very similar including the degree of buried surface area, strand swapping allows a binding reaction with a very high specificity but only millimolar affinity (20). Based on NMR titration measurements, binding of calcium decreases the K D for Ecad12 homoassociation from 10 to 0.72 mM (22). A higher K D because of a stabilization of the monomeric and/or a destabilization of the dimeric form would lower the number of adhesive contacts (20). Tcad1 did not show specific concentration-dependent NMR spectral changes that would indicate dimer formation in the presence or absence of calcium, and Tcad1-3 as well as Tcad1-5cmp showed no significant affinity for homoassociation in the solid-phase binding assays. Based on these data, the K D for T-cadherin homoassociation is estimated to be significantly larger than for Ca 2ϩ -free Ecad12 (10 mM,Ref. 22). A higher stability of monomeric Tcad1 would be in agreement with the abovedescribed structural differences to classical cadherins and with the observation that calcium-free T-cadherin is more resistant to proteolytic cleavage (28). The number of adhesion-mediating dimers should be highly dependent on concentration and increase quadratically with the cadherin surface density (20). The surface density of classical cadherins can be controlled through interactions of their cytoplasmic domain with catenins and other signaling molecules, which direct them to the cell-cell contact sites (45,46). T-cadherin does not have a cytosolic domain and localizes at lipid rafts as other GPI-anchored signaling proteins (47). This may be a further reason for the observed differences in mediating intercellular interactions.
The low adhesive capacity of T-cadherin is reminiscent of the behavior of Protocadherin-␣. For both proteins, the low homophilic capacity coincides with the replacement of Trp-2 and several other residues important for cell-cell adhesion in classical cadherins. Protocadherin-␣ is only able to adhere to HEK293T cells upon activation of ␤1 integrin by Mn 2ϩ or a specific antibody (TS2/16), which induces binding of ␤1 integrin to Protocadherin-␣ (38). Early after the discovery of T-cadherin, it was suggested that lateral association with auxiliary molecules influences its clustering on the cell surface and thereby its ability to mediate intercellular interactions (28). One may therefore speculate that similar to Protocadherin-␣ (38) interaction partners such as adiponectin (15), LDL (13,16), or yet to be identified proteins are involved in cell-cell recognition by T-cadherin.
In summary, specific amino acid replacements in T-cadherin favor a closed monomeric form over the strand-exchange dimeric form of classical cadherins. These comprise the crucial I2W replacement, as well as other substitutions, which reduce the size of the binding pocket for Ile-2, modify the backbone conformation and flexibility around residues 5 and 15, and alter charge interactions around position 2. The binding of calcium induces significant spectral changes only around the calcium binding sites. Consistent with EM and solid-phase binding data, the observed spectral changes do not give any indication of homophilic interactions. Thus T-cadherin appears to be far less adhesive than classical cadherins and, consistent with its role in cell growth and migration, is likely to be involved in reversible and dynamic cellcell adhesion-deadhesion.