Functional analysis of the structural basis of homophilic cadherin adhesion

Abstract

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.

FIGURE 1

Schematic of the cadherin monolayers used in this study.

FIGURE 2

(A) Normalized force between oriented FcCEC1-5 monolayers versus the distance between the supporting membranes. CEC1-5 dimers fused with the Fc domain were immobilized on supported protein A monolayers. The cadherin density on each membrane was 9.9 ± 0.4 × 10³ cadherin/μm². The bathing medium contained 10 mM Tris buffer, 150 mM NaNO₃, and 2 mM CaCl₂ at pH 7.0. The temperature was 25°C. Upon approach, the proteins repel at D < 600 Å. The filled circles indicate the forces measured during approach to D < 400 Å, and the open circles indicate forces measured during subsequent separation. At the position of the maximal attractive force, the protein-protein bonds yield and the surfaces jump out of adhesive contact. The filled and open triangles indicate the force curve measured during approach to 400 Å < D < 500 Å and separation, respectively. Squares show the force profile measured when the minimal separation was D > 500 Å. (B) Illustration of opposed, immobilized FcCEC1-5 monolayers.

FIGURE 3

(A) Normalized force between oriented FcCEC1-4 monolayers versus the distance between the supporting membranes. The lipid monolayer composition was as described in Fig. 1, and the solution conditions are given in the text. This truncated cadherin fragment also adhered at three different antiparallel alignments. The positions of these three minima are shifted 120 Å relative to the three minima in Fig. 2. The FcCEC1-4 surface density was 1.2 ± 0.4 × 10⁴ cadherin/μm². (B) Illustration of the opposed, immobilized FcCEC1-4 monolayers.

FIGURE 4

(A) Normalized force between oriented, immobilized FcCEC12 monolayers versus the distance between the supporting membranes. The lipid composition and solution conditions were as described in the text. The cadherin surface density was 1.7 ± 0.4 × 10⁴ cadherin/μm². These proteins adhered at a single, unique distance of 271 ± 14 Å. (B) Illustration of the corresponding immobilized FcCEC12 monolayers.

FIGURE 5

(A) Normalized force between oriented, immobilized FcCEC345 monolayers as a function of the distance between the supporting membranes. The lipid composition and solution conditions were as described in Fig. 1. The cadherin surface density was 1.2 ± 0.4 × 10⁴ cadherin/μm². This protein fragment undergoes homophilic adhesion at a single distance of 386 ± 4 Å. (B) Illustration of the corresponding immobilized FcCEC345 monolayers.

FIGURE 6

Model showing the domains required for the three adhesive minima between full-length cadherin ectodomains. The upper panel indicates the scaled energy-distance profile between FcCEC1-5 monolayers. The cartoons shown in Fig. 6 B indicate the domains required for each of the three adhesive interactions indicated in Fig. 6 A. The innermost adhesive minimum requires EC3 (black domains), the second minimum requires domains 1 and 3 (gray), and the outer minimum requires EC1 (black domains).