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First published online 23 August 2005
doi: 10.1242/jcs.02539

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Cadherin adhesion depends on a salt bridge at the N-terminus

The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK

View larger version (37K): [in a new window]   Fig. 1. The salt bridge between Asp1 and Glu89. (a) Strand exchange in the C-cadherin structure 1L3W. The two domain 1 protomers are shown in blue and yellow respectively and Trp2 is coloured purple. Juxtaposition of the nitrogen of the N-terminal amino group of Asp1 (blue) and the oxygen atoms of the acidic side chain of Glu89 (red) are shown. (b) Cell surface staining of mutant and wild-type (Wt) N-cadherins expressed by K562 transfectants, matched for equal expression. The mutations Glu89Ala (E89A) and Gly-Gly (GG; in which the N-terminus was extended by two glycine residues) were present singly or together in the same molecule. The shaded profile is a negative control using FITC-labelled second antibody only.

View larger version (16K): [in a new window]   Fig. 2. Effect of the salt bridge on cadherin-mediated cell adhesion. (a) Adhesion of N-cadherin transfectants to wild-type or mutant N-cadherin Fc fusion proteins coated at 1 µg/ml. Extension of the N-terminus by two amino acids (GG) or the mutation Glu89Ala (E89A) inhibited adhesion to wild-type N-cadherin but, when present in opposing molecules, they formed a complementary pair resulting in enhanced adhesion. The mutation Asp134Ala (D134A) prevents coordination of calcium in the junction between domains 1 and 2 and served as a negative control. (b) Adhesion between Glu89Ala and the Gly-Gly extension mutant was ablated by the mutation Asp134Ala. (c) Enhanced adhesion between the complementary pair, Gly-Gly and Glu89Ala, compared with adhesion between wild-type N-cadherin molecules over a range of concentrations of the Fc fusion proteins.

View larger version (15K): [in a new window]   Fig. 3. Binding of soluble N-cadherin Fc to cell surface N-cadherin. Mutant or wild-type Fc fusion proteins were used to `stain' cell surface N-cadherin expressed by K562 transfectants. Binding was detected with FITC-labelled anti-Fc. The shaded profiles are negative controls using N-cadherin Fc with the mutation Asp134Ala. Results show that the affinity between wild-type cadherin molecules was too low to give detectable binding, whereas the interaction between Glu89Ala (E89A) and the Gly-Gly mutant (GG) gave strong staining.

View larger version (13K): [in a new window]   Fig. 4. Effect of the prodomain. L cells expressing N-cadherin with an uncleaved prodomain were tested for adhesion to mutant or wild-type N-cadherin Fc fusion proteins. (a) L cells adhered to the Glu89Ala (E89A) mutant but not to wild-type N-cadherin. (b) Western blot demonstrating removal of the prodomain by treatment of the L cell transfectants with trypsin. (c) After treatment, the L cells adhered strongly to wild-type N-cadherin whereas adhesion to the Glu89Ala mutant was greatly diminished.

View larger version (39K): [in a new window]   Fig. 5. Proposed role of the salt bridge in the strand-swap mechanism in mutant and wild-type N-cadherin. A full explanation is offered in the text. (a) Domain 1 in isolation. An equilibrium between docked and undocked Trp2 favours the docked form because the salt bridge between Glu89 and the N-terminus (shown as a dotted line) stabilises Trp2 insertion. (b) Adhesion between wild-type molecules. A transition state in which Trp2 is undocked is sampled from either side and is depicted as an `unshaded' tryptophan. Adhesion is moderate. (c) The salt bridge on one side is prevented by extension of the N-terminus. (d) The salt bridge is prevented by the mutation Glu89Ala. In both c and d, adhesion is very weak. Although one strand can cross-intercalate, the process competes unfavourably with intramolecular docking of Trp2 into the wild-type domain. (e) The two mutations form a complementary pair. Intramolecular docking is prevented and therefore the activation barrier for strand exchange is lowered. Exchange of one strand is possible and cross-intercalation of Trp2 is stabilised by the salt bridge. Adhesion is enhanced. (f) The double mutation is present on each side. The salt bridge cannot form to support strand exchange so there is no adhesion.

View larger version (38K): [in a new window]   Fig. 6. Removing Trp2 or blocking the hydrophobic acceptor pocket. (a) K562 cells expressing wild-type or mutant N-cadherin were tested for adhesion to N-cadherin Fc (1 µg/ml) bearing either the mutation Trp2Gly (W2G) or the pocket-blocking mutation Ala80Ile (A80I). The Trp2Gly mutant acted as a strand acceptor and therefore adhered to cells expressing the Glu89Ala (E89A) mutation. In contrast, the Ala80Ile mutant behaved as a strand donor because intramolecular docking of Trp2 was denied and therefore bound to cells expressing the Gly-Gly N-terminal extension mutant (GG). (b) Dynabeads coated with the Trp2Gly mutant or the Ala80Ile mutant were tested for aggregation separately or as a mixture and compared with aggregation mediated by wild-type N-cadherin. Aggregation was assessed by microscopy. The Asp134Ala (D134A) mutant served as a negative control and here, the 2.8 µm diameter beads remained monodisperse.