Mechanism of E-cadherin dimerization probed by NMR relaxation dispersion

Significance

Dimerization of transmembrane protein epithelial cadherin (E-cadherin) molecules extending from apposing cells is a critical event in calcium-dependent cell adhesion, a process essential for the formation and maintenance of tissues and organs. This study elucidates the molecular mechanism of E-cadherin dimerization by utilizing nuclear magnetic resonance spectroscopy to detect and characterize sparsely populated molecular species. In this mechanism, E-cadherin monomers first interact at an auxiliary interface to form a dimeric intermediate, which is followed by the conformational changes required to form the mature dimer. Besides contributing to the understanding of E-cadherin binding and recognition, the mechanism described in this work may illustrate a more general mechanism by which proteins with occluded binding sites interact with each other.

Abstract

Epithelial cadherin (E-cadherin), a member of the classical cadherin family, mediates calcium-dependent homophilic cell–cell adhesion. Crystal structures of classical cadherins reveal an adhesive dimer interface featuring reciprocal exchange of N-terminal β-strands between two protomers. Previous work has identified a putative intermediate (called the “X-dimer”) in the dimerization pathway of wild-type E-cadherin EC1–EC2 domains, based on crystal structures of mutants not capable of strand swapping and on deceleration of binding kinetics by mutations at the X-dimer interface. In the present work, NMR relaxation dispersion spectroscopy is used to directly observe and characterize intermediate states without the need to disrupt the strand-swapped binding interface by mutagenesis. The results indicate that E-cadherin forms strand-swapped dimers predominantly by a mechanism in which formation of a weak and short-lived X-dimer–like state precedes the conformational changes required for formation of the mature strand-swapped dimeric structure. Disruption of this intermediate state through mutation reduces both association and dissociation rates by factors of ∼10⁴, while minimally perturbing affinity. The X-dimer interface lowers the energy barrier associated with strand swapping and enables E-cadherins to form strand-swapped dimers at a rate consistent with residence times in adherens junctions.

Epithelial cadherin (E-cadherin) is a type I cadherin in the classical cadherin family. E-cadherin mediates calcium-dependent homophilic cell adhesion and plays important roles in the formation and maintenance of tissues and organs (1). E-cadherin consists of an ectodomain composed of five similar tandemly repeated extracellular cadherin-like (EC) domains connected by conserved calcium-binding linker regions, followed by a single transmembrane segment, and an intracellular region. The ectodomain mediates assembly into adherens junctions (2), which are intercellular structures that maintain physical association between cells (3). The self-assembly of adherens junction is a cooperative process involving both homodimerization in trans (between cadherins from different cell surfaces) and lateral oligomerization in cis (between cadherins from the same cell surface) (4, 5).

The N-terminal outermost extracellular domain (EC1) is directly involved in the trans association of E-cadherins from apposing cells and dictates the specificity of cell adhesion. Crystal structures of extracellular domains reveal a dimer interface characterized by reciprocal exchange of the N-terminal half of the first β-strand in EC1, termed the A*-strand, from a pair of protomers (Fig. 1 A and B) (6). In type I cadherins, a single conserved and functionally important residue Trp2 acts as an anchor residue for exchange of the A*-strand across the dimer interface. Monomers assume a closed conformation with Trp2 and the A*-strand docked in cis into a hydrophobic pocket on the same monomer. In dimers, protomers assume a strand flipped-out “open” conformation with the hydrophobic pocket available for structurally similar binding, but in trans, with the partner molecule. The strand-swapped interface has been observed for all members of classical cadherin family (7) and is essential for adhesive activity. Strand exchange is an example of “3D domain swapping” (8) and serves as the structural basis for the low but differential association constants critical for the specificity of cell adhesion (9).

Fig. 1.

Crystal structures of E-cadherin EC1–EC2 domains. (A) Strand-swapped dimer of wild-type [Protein Data Bank (PDB) ID code 2qvf]. (B) Close-up view of the strand-swapped dimer interface of wild type, and (C) X-dimer of E89A mutant (PDB ID code 3lni). The green spheres represent bound calciums.

An alternative dimer interface was identified from crystal structures of site-directed mutants of E-cadherin designed to abolish strand swapping (10). Highly similar X-dimer structures, so named according to the shape of the complexes, were obtained for W2A and E89A site-directed mutants and a variant with an Ala-Ala extension at the N terminus. The X-dimer interface centers around the linker region between the first and second EC domains (Fig. 1C), does not involve strand swapping, and does not overlap substantially with the strand-swapped interface. NMR (11) and single-molecule FRET (12) studies on mutants not capable of strand swapping have detected transiently formed complexes, presumably the X-dimers, with association constants significantly lower than those of the strand-swapped dimers of wild-type E-cadherin. At the cellular level, a recent study has suggested a role of the X-dimer interface in the disassembly of adherens junctions (13).

Our previous work has shown that the K14E mutation, which destabilizes the X-dimer interface by breaking the salt bridge between the K14 side-chain amino group and D138 main-chain carbonyl group, results in significantly slower binding kinetics than wild-type E-cadherin and loss of cell adhesion in cell aggregation assays (10). However, the K14E mutant forms the same strand-swapped dimers with similar dissociation constants as the wild-type protein. The M188D mutant of the type II cadherin 6 also displays slower binding kinetics, owing to disruption of the X-dimer interface (10). These results suggest that formation of the X-dimers is a critical step in the dimerization pathway of at least some classical cadherins.

All extant structural evidence for an X-dimer intermediate relies on mutant proteins incompetent for strand swapping. The ability to observe sparsely and transiently populated states by NMR spectroscopy allows detection of intermediate states in the dimerization of E-cadherin without the need to disrupt the primary, strand-swapped binding interface by mutagenesis. In this work, we directly observed and characterized an intermediate state, consistent with the X-dimer configuration, in the binding pathway of wild-type E-cadherin using NMR relaxation dispersion spectroscopy. We have quantified the binding kinetics of both wild-type and the K14E mutant of E-cadherin, demonstrating that the X-dimer interface controls both the association and dissociation kinetics of E-cadherin with minimal effects on affinity. The binding of wild-type E-cadherin on a ∼1-s timescale is consistent with the fast turnover of E-cadherin in adherens junctions; the loss of cell adhesion in the K14E mutant indicates that the kinetic rate constants of the K14E mutant measured in solution are likely the lower limit for the normal function of E-cadherin.

Recent NMR studies have shed light on the molecular details of recognition and binding between protein–ligand or protein–protein partners that differ in free and bound conformations, especially in the case of coupled binding and folding of intrinsically disordered proteins (14, 15). Dimerization of E-cadherin represents a case in which binding can occur through initial interactions at an auxiliary interface, without first requiring the exposure of the primary binding site. Besides elucidating the dimerization mechanism of E-cadherin, this work demonstrates the utility of NMR relaxation dispersion techniques in understanding the mechanisms of binding between protein molecules with occluded binding sites, which are largely unknown due to the difficulty in defining structural characteristics of kinetically important intermediate states.

Results

Binding Kinetics of Wild-Type E-cadherin and K14E Mutant.

Wild-type E-cadherin EC1–EC2 domains (ECAD12) form strand-swapped dimers in solution and the monomeric and dimeric states are in slow exchange on the NMR chemical shift timescale (Fig. S1). The dissociation constant obtained by quantifying peak volumes in ¹H-¹⁵N heteronuclear single-quantum coherence (HSQC) spectra is 82 ± 13 μM, which agrees with a dissociation constant of 99 ± 16 μM previously determined from analytical ultracentrifugation (AUC) experiments (16). Bimolecular on- and off-rates of wild-type ECAD12 were measured using ¹⁵N ZZ-exchange experiments for a sample containing 105 μM free monomers and 134 μM dimers. Composite ratios constructed from the intensities of well-resolved exchange and autocorrelation peaks were fit to a quadratic function of the mixing time (SI Text). Fig. 2A shows the composite ratio Ξ(T_mix) as a function of mixing time for two selected residues. The best-fit value for 4k_off k_on [M] is 3.6 ± 0.5 s^–2, where [M] is the concentration of monomers. Using the known dissociation constant (82 μM), the on- and off-rate constants were calculated to be k_on = (1.0 ± 0.1) × 10⁴ M^–1⋅s^–1 and k_off = 0.8 ± 0.1 s^–1, respectively, at 299 K. The off-rate agrees with a value of 0.7 s^–1 reported by a previous NMR study of an ECAD12 construct containing six additional residues at the C terminus (17).

Fig. 2.

(A) Buildup curve of the composite peak intensity ratio Ξ(T_mix), which reflects the exchange rate between monomer and dimer states of wild-type ECAD12. The Inset shows ¹⁵N ZZ-exchange spectra of residue I38 at several mixing times. (B) The time course of fluorescence intensity of K14E mutant in a rapid dilution experiment. The solid lines are best fits to the experimental data.

We have previously shown that the K14E mutation, which breaks the salt bridge between K14 and D138 at the X-dimer interface, significantly slows dimerization kinetics. Formation of dimers by the mutant protein can be detected by long-time frame AUC experiments, but not by short-time frame surface plasmon resonance (SPR) experiments. Nevertheless, the K14E mutant forms native strand-swapped dimers, as confirmed by the crystal structure (10). The dissociation constant of the K14E mutant determined by NMR spectroscopy is 104 ± 11 μM and agrees with the value of 117 ± 8 μM previously determined by AUC. We quantified the binding kinetics of the K14E mutant by monitoring the time course of dimer dissociation after rapid dilution by intrinsic tryptophan fluorescence spectroscopy and ¹H-¹⁵N transverse relaxation-optimized spectroscopy (TROSY). Fig. 2B shows the time dependence of fluorescence intensity. The fluorescence-based experiment has much higher sensitivity and allows simpler data analysis than the corresponding NMR experiment (Fig. S2C). A control fluorescence experiment was performed on wild-type ECAD12 to account for effects of photobleaching (Fig. S2B and SI Text). Fitting the data to an integrated first-order rate equation yielded k_off = (2.1 ± 0.2) × 10⁻⁴ s^–1. Taking into account the 10% underestimation of the rate constant due to photobleaching, the corrected k_off is 2.3 × 10⁻⁴ s^–1. Using the known dissociation constant (104 µM), k_on was calculated to be 2.2 M^–1⋅s^–1. Thus, the binding kinetics of the wild-type and the K14E mutant differ by approximately four orders of magnitude despite their similar binding affinities.

A Dimeric Intermediate State Detected by ¹H R₂ Relaxation Dispersion Experiments.

Chemical exchange processes on microsecond-to-millisecond timescales can be effectively detected and characterized by transverse relaxation dispersion NMR spectroscopy. In these experiments, kinetic, thermodynamic, and structural information on the exchange process is obtained by measuring transverse relaxation rate, R₂, as a function of the number of 180° pulses, evenly spaced within a constant relaxation time period (18, 19). Amide ¹⁵N relaxation dispersion has been extensively used to probe chemical exchange processes; however, preliminary measurements indicated that the amide ¹H is a more sensitive probe of exchange processes in E-cadherin. Accordingly, ¹H relaxation data were recorded on wild-type ECAD12 at two protein concentrations, 374 and 97 μM (as represented by the total concentration of monomeric units), using TROSY-selected ¹H Carr–Purcell–Meiboom–Gill (CPMG) experiments. The high and low concentration samples contain 105 and 46 μM free monomers, respectively, at equilibrium. Relaxation dispersion curves for all assigned resonances with sufficient sensitivity are shown in Fig. S3.

Four residues show substantial (>5 s^–1) ¹H relaxation dispersion, including I7m, E11, K14m, and Q101m (Fig. 3A), where “m” denotes the monomeric state. For E11, monomer and strand-swapped dimer cross-peaks are degenerate and cannot be distinguished. The relaxation dispersion profiles are concentration dependent, suggesting that the minor conformational state is dimeric with a population that depends on protein concentration. Data recorded at 600- and 800-MHz ¹H frequencies for the high-concentration sample were globally fit to a two-site exchange model (Table S1 and Fig. S4). K14m was excluded from fitting due to the large uncertainties in the measured relaxation rates. The best-fit values for the global parameters are k_ex = 1,890 ± 130 s^–1 and p_m = 0.025 ± 0.003, where k_ex is the sum of forward pseudo first-order and reverse kinetic rate constants and p_m is the population of the minor conformational state. Fitting individual residues independently does not statistically improve the fit according to an F test. Best-fit values for k_ex and Δω_H, which is the difference in amide ¹H chemical shifts between the major and minor conformational states, determined from the high-concentration sample were used to fit the data recorded on the low-concentration sample at 600 MHz and yielded p_m = 0.017 ± 0.003.

Fig. 3.

(A) ¹H relaxation dispersion profiles of wild-type ECAD12 at two different protein concentrations and K14E mutant (blue). The “m” denotes the monomeric state. The total monomer concentrations were 374 μM (red) and 97 μM (purple) for two wild-type samples, respectively; the concentration of the K14E mutant was 375 μM. τ_cp is the spacing between 180° pulses. For clarity, only data recorded at 600 MHz are shown. (B) X-dimer interface with residues showing relaxation dispersion highlighted by stick representation. The green spheres represent bound calciums.

All four residues showing significant dispersion are located at or close to the X-dimer interface defined by X-ray crystallography (Fig. 3B). K14 and Q101 are directly involved in key contacts at the X-dimer interface: K14 side chain forms a salt bridge with the main-chain carbonyl group of D138, whereas the Q101 side chain forms hydrogen bonds with the D100 main chain and the N143 side chain. D138 and N143 are located at or close to the BC loop comprising residues 136–140 in the EC2 domain. Several residues in this loop and neighboring regions show broadened ¹⁵N and ¹H resonances (Fig. S5), indicative of microsecond-to-millisecond timescale chemical exchange processes, but signal intensities were too low to permit determination of kinetic parameters. The crystal structure reveals an additional salt bridge between R105 and E199; however, these two residues do not show relaxation dispersion. This difference could reflect stabilization of the X-dimer either by crystal packing forces or the mutations necessary to abolish strand swapping.

Residues E11 and I7 are not involved in direct contacts with the other protomer. However, E11 is spatially close to the linker region and, together with Q101, binds to one of the three calcium ions located at the linker region. E11 has been suggested to play a role in strand swapping by serving as one of the two anchor points of the short A/A* strand, introducing conformational strain in the monomer structure, and enhancing the relative stability of the strand-swapped dimer conformation (20). I7 is located right after two prolines, P5 and P6, that serve as the hinge connecting the A*-strand with the remaining portion of the A-strand. Formation of X-dimers results in large perturbations in the I7 ¹H chemical shift, Δω_H = 0.45 ppm, as determined from ¹H relaxation dispersion data, but not in the ¹⁵N chemical shift, Δω_N ≈ 0, as indicated by lack of significant dispersion in ¹⁵N experiments. These results suggest that chemical exchange line broadening reflects changes in the local environment, such as positioning of aromatic rings, due to X-dimer formation, rather than dihedral angle or other conformational changes, because Δω_H >> Δω_N. In contrast, in the strand-swapped dimers, ¹⁵N is shifted downfield by 2.28 ppm and ¹H is shifted downfield by 0.30 ppm with respect to the monomer resonances. These results are consistent with conformational changes, because Δω_H ≈ Δω_N, measured in angular frequency units. Overall, the residues showing relaxation dispersion or line broadening overlap with those located at the X-dimer interface identified from crystal structures (Fig. S6), indicating that the minor conformational state is structurally similar to the X-dimer.

The Intermediate State Is Undetectable in the K14E Mutant.

To test whether the K14E mutation affects the minor conformational state observed in the wild-type samples, we performed ¹H CPMG experiments on the mutant under the same conditions. The K14E mutant sample contained 116 μM free monomers and 129 μM dimers. None of the residues undergoing exchange processes in wild-type ECAD12 shows relaxation dispersion in the K14E mutant (Fig. 3A), indicating that either the population of the minor conformational state is below the detection limit of ∼0.5% or the exchange process shifts to an NMR chemical shift timescale too fast to yield detectable line broadening. Changes in timescale can result from either smaller chemical shift differences between exchanging states or increases in the kinetic exchange rate, which is predominated by the dissociation rate of the X-dimer. Both smaller perturbations in chemical shifts and higher dissociation rates correspond to weaker interactions and lower stability of the minor conformational state. Overall, the results indicate that the salt bridge between K14 and D138, observed in the crystal structure, is also essential for the stability of the minor conformational state observed by NMR, providing additional support for the conclusion that the minor state is highly similar to the X-dimer crystal structures.

Kinetic Model for ECAD12 Association and Dissociation.

A two-step model has been proposed for E-cadherin dimerization based on the qualitative difference in binding kinetics of wild-type and K14E mutant ECAD12, detected by AUC and SPR experiments (10). In this model, monomers interact bimolecularly to form a hypothetical X-dimer intermediate followed by a unimolecular rearrangement to form the strand-swapped dimer. The current study elaborates on this model by detecting and characterizing the X-dimer intermediate state using relaxation dispersion NMR experiments. Combined with ZZ-exchange NMR experiments, the kinetic parameters were determined at bulk equilibrium.

Without loss of generality, ECAD12 dimerization can be represented by the kinetic scheme shown in Fig. 4. This scheme consists of the X-dimer–dependent pathway and an alternative pathway collectively representing any other, possibly multistep, binding mechanisms. The ∼10⁴ reduction in the kinetic rate constants for the K14E mutant, in which formation of X-dimer is at least partially abrogated, compared with wild-type ECAD12, indicates that X-dimer–dependent pathway is the major if not the only pathway under our experimental conditions. We note that, for bimolecular reactions involving multiple pathways and mechanisms, the relative fluxes through each pathway are generally concentration dependent (21).

Fig. 4.

Pathways of ECAD12 dimerization. M denotes monomers, X-D denotes X-dimer–like intermediates, and SS-D denotes strand-swapped dimers.

In the major pathway, under steady-state conditions, the overall association rate constant is as follows:

The ¹H relaxation dispersion experiments on the wild-type sample at 374 μM yielded 2k₁[M] + k₋₁ = k_ex = 1,890 s⁻¹, 2k₁[M]/k_ex = p_m = 0.025. The ZZ-exchange experiment yielded k_on = 1.0 × 10⁴ M⁻¹⋅s^–1 and k_off = 0.8 ± 0.1 s^–1. Combining these results gives k₁ = 2.3 × 10⁵ M^–1⋅s^–1, k_–1 = 1.84 × 10³ s^–1, and k₂ = 86 s^–1. The value of k₁ falls into the 10⁵ to 10⁶ M^–1⋅s^–1 range of the theoretically estimated bimolecular on-rates for protein–protein association in the absence of long-range forces (22). Because k_–1 ≫ k₂,

namely, the bimolecular on-rate of the overall binding reaction is proportional to the association constant of the intermediate state, K_I = k₁/k_–1, and the rate of conversion from X-dimers to strand-swapped dimers, k₂. For the dissociation pathway, k_–2 defines the rate-limiting step; therefore, k_–2 ≈ k_off = 0.8 s⁻¹.

Fig. 5 shows free-energy diagrams for dimerization of wild-type and K14E mutant ECAD12. No intermediate state was observed experimentally by relaxation dispersion for the K14E mutant, confirming that this mutation decreases K_I by disrupting the K14-D138 salt bridge. In addition, the drastic reduction in both k_on and k_off without significant change in affinity of the K14E mutant suggests that the K14E mutation perturbs the transition state associated with the conversion of X-dimers to strand-swapped dimers with minimal effects on the structures and relative stability of monomers and strand-swapped dimers. As evident from Fig. 5, the transition state must be perturbed by the mutation in order for k_–2 to decrease, whereas the change in k₂ relies on the change in the relative free energy of the X-dimer and the transition state. Destabilization of the intermediate state and elevation of the energy of the transition state collectively contribute to the nearly four orders of magnitude change in k_on, whereas the change in k_off solely results from decrease in k₋₂. The approximate expression for dissociation constant is K_d = k_off /k_on ≈ (1/K_I)(k₋₂/k₂). Destabilization of X-dimer has the exactly opposite effects on 1/K_I and k_–2/k₂, and therefore no net effect on K_d, consistent with the nearly unchanged K_d of the K14E mutant.

Fig. 5.

The free-energy diagrams of (A) wild type and (B) K14E mutant of ECAD12 at 299 K and 1 M standard concentration. M represents monomeric state, X-D represents X-dimer–like intermediate state, SS-D represents strand-swapped dimeric state, and TS represents transition state.

For wild-type ECAD12, the binding reaction proceeds in two steps via major pathway shown in the scheme in Fig. 4. The kinetically favorable X-dimer state accumulates, because the activation energy for the second step is larger than that for the essentially diffusion-controlled first step. The molecular mechanism of diffusion-controlled protein–protein association has been extensively investigated by both experimental and computational studies (22, 23). Long-range electrostatic interactions contribute to the on-rate (24, 25) and short-range interactions, including hydrophobic and van der Waals interactions, hydrogen bonds, and salt bridges, dictate the off-rate. The conformational changes associated with strand swapping occur during the rate-limiting unimolecular second step. For the K14E mutant, neither the binding mechanism nor the rate constants of each elementary reaction are known. Therefore, the free-energy diagram (Fig. 5B) shows a single overall activation barrier for simplicity, although intermediate states could exist at levels undetectable by the relaxation dispersion experiments.

Discussion

We have used ¹H relaxation dispersion and ¹⁵N ZZ-exchange NMR spectroscopy to investigate the pathway of E-cadherin trans association. The studies were conducted on an E-cadherin construct containing the EC1 and EC2 domains and an intact calcium-binding linker region between the two domains. We observed significant relaxation dispersion for residues that lie at the X-dimer interface, resulting from millisecond timescale chemical exchange processes between monomers and the dimeric intermediate state. In contrast, Trp2 side-chain Hε resonance does not show detectable relaxation dispersion, suggesting that it adopts a conformation similar to the monomer state, i.e., a closed conformation, in the intermediate state. This work has provided direct evidence for a dimeric intermediate state, with key structural features consistent with crystal structures of X-dimers, in the dimerization pathway for wild-type E-cadherin. It also uncovered a mechanism by which the kinetic barrier associated with strand swapping, or in general, 3D domain swapping, is lowered. In this mechanism, an auxiliary interface is used to form a short-lived dimeric intermediate state, which leads to an energetically favorable transition state that partially resembles the intermediate state and accelerates overall binding kinetics. Formation of the X-dimer state leads to changes in the conformational energy landscape, which lower the effective activation barrier associated with strand swapping by localizing the two β-strands in close proximity. A key factor in this mechanism is that the bimolecular intermediate state needs to be formed with high enough association constant; otherwise, the low population of the intermediate state becomes the kinetic bottleneck of the overall reaction (26). This factor is especially a problem for proteins with occluded binding sites. In the case of E-cadherin, the X-dimer interface allows formation of a dimeric intermediate state that is thermodynamically stable enough for fast overall binding kinetics, yet not so stable as to prevent the formation of the final strand-swapped dimers. The X-dimer interface does not overlap substantially with the strand-swapped dimer interface, which may allow tuning of binding kinetics without affecting the association constant of strand-swapped dimers.

Although the current data mainly support an X-dimer–dependent mechanism for wild-type E-cadherin, other mechanisms cannot be completely excluded, because binding is not completely kinetically prohibited for the K14E mutant. The on- and off-rates of the K14E mutant represent the upper limit of the kinetic constants of alternative pathways, although the exact binding mechanisms are not known for these minor pathways. Previously, a monomeric and A*-strand flipped-out state with a population of 0.032 ± 0.004 was detected and characterized in a construct of mouse type II cadherin 8 containing only the EC1 domain, using ¹⁵N relaxation dispersion and solvent exchange measurements (27). The presence of this sparsely populated monomeric “open” species suggests an alternative binding mechanism, at least for cadherin 8, in which two “open” monomers collisionally interact to form the strand-swapped dimer. However, because the cadherin 8 construct is incapable of forming X-dimer state owing to the lack of an intact linker region and the EC2 domain, the importance of this mechanism in the context of wild-type protein consisting of at least EC1 and EC2 domains, as studied herein, is not known. No relaxation dispersion was observed for side-chain Nε and Hε resonances of Trp2 in ECAD12 monomers, suggesting that the populations of any “flipped-out” states of ECAD12 are below the limit of detection.

E-cadherin is a major component of adherens junctions, which are constantly renewing structures with individual cadherin molecules entering and exiting the junctions (28). Understanding the molecular mechanisms underlying the turnover of cadherins in the adherens junctions requires knowledge about not only thermodynamics but also kinetics of cadherin association and dissociation. The apparent half-residence times of E-cadherins in adherens junctions have been determined by fluorescence photobleaching experiments to range from 50 s to 4 min depending on the type of cells used for the measurements (2, 29). The ∼1-s timescale for E-cadherin association and dissociation in solution allows the trans dimers to form within the lifetime of E-cadherins in junctions; consequently, strand swapping does not present a kinetic barrier for the cadherin turnover. The resulting favorable kinetic properties allow strand-swapped dimers to form and dissociate on a timescale required for physiological functions.

The measurements described here are carried out in 3D bulk solution, whereas intercellular cadherin dimerization occurs in the quasi-2D environment of a cell surface. We have previously derived an equation that relates 3D to 2D binding affinities (30, 31). A critical parameter is the thickness of the reaction shell between the two cell membranes that defines a region accessible to the EC1 domains of cadherins located on apposed membrane surfaces (31). The 2D affinity of E-cadherin is ∼100 μm^–2 and is in the range of cadherin densities on cell surfaces (9). Thus, the experimental conditions used in the current work, where concentrations are in the range defined by the 3D affinity approximate the conditions on cell surfaces.

Conclusions

We have defined the dimerization pathway of E-cadherin extracellular domains EC1–EC2 by NMR and intrinsic Trp fluorescence spectroscopy. An intermediate state with structural features consistent with previously determined X-dimer structures was detected and characterized by ¹H R₂ relaxation dispersion experiments. Mutation of the key residue K14 at the X-dimer interface to E, disrupting the salt bridge between K14 and D138, decreases the population of the intermediate to an undetectable level, consistent with the destabilizing effects of the mutation, and dramatically reduces both forward and reverse binding kinetics compared with wild-type protein. These results support an X-dimer–dependent dimerization pathway and define the rate constants of the elementary reactions. The existence of X-dimer interface accelerates otherwise extremely slow association and dissociation kinetics, exhibited by the K14E mutant, allowing E-cadherins to form trans dimers at a rate consistent with residence times in the adherens junctions. Applying these experimental methods to other members of cadherin family will lead to better understanding of the general mechanisms of cadherin binding and recognition.

Materials and Methods

NMR Samples.

The constructs contain 1–213 residues of the mature form of mouse E-cadherin. Experimental details on protein expression and purification have been included in SI Text. The NMR buffer comprises 5% (vol/vol) D₂O/95% (vol/vol) H₂O, 10 mM CaCl₂, 120 mM NaCl, 10 mM Tris⋅HCl, pH 7.9. Protein concentration was determined by absorbance at 280 nm using an extinction coefficient of 21,430 M^–1⋅cm^–1.

NMR Spectroscopy.

All NMR experiments were performed at 299.2 K unless stated otherwise and on Bruker 600-, 800-, and 900-MHz spectrometers equipped with triple-resonance z-gradient cryogenic probes. d6-methanol was used to calibrate sample temperature (32). Backbone resonance assignments were obtained using triple-resonance correlation experiments with a TROSY detection scheme, including HNCO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB, together with 3D ¹⁵N-edited NOESY-TROSY (33), on a sample of ²H-, ¹³C-, ¹⁵N-labeled wild-type ECAD12 containing 0.8 mM protein. Additional experimental details have been included in SI Text.

The relative quantity of monomers and dimers for a given sample was determined from the ratio of peak volumes in ¹H-¹⁵N HSQC spectra acquired with 6-s recycle delay. The ratio was corrected for the differential loss of magnetization for monomers and dimers during magnetization transfer periods in the sequence according to a previously reported method (34).

The pulse sequence for ¹⁵N ZZ-exchange experiment is depicted in Fig. S7. The experiments were performed on a sample containing 374 μM ²H-, ¹⁵N-labeled wild-type ECAD12 at 600 MHz (see SI Text for additional experimental details).

The pulse sequence for ¹H CPMG experiments is depicted in Fig. S8. Data were recorded on two ²H-, ¹⁵N-labeled wild-type ECAD12 samples at two protein concentrations, 374 and 97 μM, respectively, and one ²H-, ¹⁵N-labeled K14E sample containing 375 μM protein. For the 374 μM wild-type sample, data were recorded at 600- and 800-MHz ¹H frequencies. For other samples, data were recorded only at 600-MHz ¹H frequency. The experiments were performed with constant relaxation periods of 40 and 20 ms (see SI Text for additional experimental details).

Fluorescence Experiments.

The experiments were performed at 299 K in buffers containing 10 mM CaCl₂, 120 mM NaCl, 10 mM Tris⋅HCl, pH 7.9, identical to those for NMR experiments. The excitation wavelength was 295 nm, and the detection wavelength was 330 nm. The initial concentration of the K14E sample was 223 μM and the sample was diluted to a final concentration of 3 μM rapidly and the fluorescence intensity was recorded every 10 s with 1-s sampling time for 6 h. The experiment was repeated once with a lower final concentration, 2 μM. A control experiment was performed on a wild-type sample containing 306 μM protein, which was rapidly diluted to 3 μM.