Summary Adherens junctions, which play a central role in intercellular adhesion, comprise clusters of type I classical cadherins that bind via extracellular domains extended from opposing cell surfaces. We show that a molecular layer seen in crystal structures of E- and N-cadherin ectodomains reported here and in the C-cadherin structure corresponds to the extracellular architecture of adherens junctions. In all three ectodomain crystals, cadherins dimerize through a trans adhesive interface and are connected by a second, cis, interface. Assemblies formed by E-cadherin ectodomains coated on liposomes also appear to adopt this structure. Fluorescent imaging of junctions formed from wild-type and mutant E-cadherins in cultured cells confirm conclusions derived from structural evidence. Mutations that interfere with the trans interface ablate adhesion, whereas cis interface mutations disrupt stable junction formation. Our observations are consistent with a model for junction assembly involving strong trans and weak cis interactions localized in the ectodomain.

We present a new method for determining the orientation of alpha-helical sections of proteins or peptides in membrane. To apply this method, membranes containing proteins must be prepared in a multilayer array. Circular dichroism (CD) spectra of the multilayer sample are then measured at the normal as well as oblique incident angles with respect to the bilayer planes; we call such spectra oriented circular dichroism (OCD). The procedure of OCD measurement, particularly the ways to avoid the spectral artifacts due to the effects of dielectric interfaces, linear dichroism and birefringence, and the method of data analysis are described in detail. To illustrate the method, we analyze the OCD of alamethicin in diphytanoylphosphatidylcholine multilayers. We conclude unambiguously that the helical section of alamethicin is parallel to the membrane normal when the sample is in the full-hydration state, but the helical section rotates to the plane of membrane when the sample is in a low-hydration state. We also obtained the parallel and perpendicular CD spectra of alpha-helix, and found them to be in agreement with previous theoretical calculations based on the exciton theory. These spectra are useful for analyzing protein orientations in future experiments.

A variety of amphiphilic helical peptides have been shown to exhibit a transition from adsorbing parallel to a membrane surface at low concentrations to inserting perpendicularly into the membrane at high concentrations. Furthermore, this transition has been correlated to the peptides' cytolytic activities. X-ray lamellar diffraction of diphytanoyl phosphatidylcholine-alamethicin mixtures revealed the changes of the bilayer structure with alamethicin concentration. In particular, the bilayer thickness decreases with increasing peptide concentration in proportion to the peptide-lipid molar ratio from as low as 1:150 to 1:47; the latter is near the threshold of the critical concentration for insertion. From the decreases of the bilayer thickness, one can calculate the cross sectional expansions of the lipid chains. For all of the peptide concentrations studied, the area expansion of the chain region for each adsorbed peptide is a constant 280 +/- 20 A2, which is approximately the cross sectional area of an adsorbed alamethicin. This implies that the peptide is adsorbed at the interface of the hydrocarbon region, separating the lipid headgroups laterally. Interestingly, the chain disorder caused by a peptide adsorption tends to spread over a large area, as much as 100 A in diameter. The theoretical basis of the long range nature of bilayer deformation is discussed.

Whereas the barrel-stave configuration is accepted by most investigators as a good description of the conducting state of alamethicin, there are conflicting interpretations on its nonconducting state; in the absence of an applied field, some found alamethicin molecules on the membrane surface, but others found them incorporated in the hydrophobic core of the membrane. This problem is resolved by the discovery of a phase-transitionlike behavior of alamethicin in the membrane. As a function of lipid/peptide ratio L/P and the chemical potential of water mu, alamethicin molecules were observed to switch between two states: in one, the majority of the peptide molecules bind parallel to the membrane surface; in another, the majority of the peptide molecules insert perpendicularly into the membrane. The state of alamethicin was monitored by the method of oriented circular dichroism (OCD; Wu, Y., H. W. Huang, and G. A. Olah, 1990, Biophys. J. 57:797-806) using aligned multilayer samples in the liquid crystalline L(alpha) phase. If L/P exceeds a critical value, most of the peptide molecules are on the membrane surface. If L/P is below the critical value, most of the peptide molecules are incorporated in the membrane when mu is high; when mu is low, most of them are again on the membrane surface. In a typical conduction experiment of voltage dependence, alamethicin molecules are in a partition equilibrium between the aqueous phase and the lipid phase before the application of voltage; in the lipid phase, the lipid/peptide ratio is such that most of alamethicin molecules are on the membrane surface. This is the nonconducting state of alamethicin. The OCD analysis showed that there is essentially no change in the secondary structure when alamethicin changes between the surface state and the inserted state. The voltage-gating mechanism can be explained if we assume that these surface peptide molecules probabilistically turn into the membrane core to form channels due to the dipole-electric field interactions. We speculate that the phase-transitionlike behavior is a manifestation of membrane-mediated intermolecular interactions between peptide molecules.

Membrane-bound receptors often form large assemblies resulting from binding to soluble ligands, cell-surface molecules on other cells, and extracellular matrix proteins1. For example, the association of membrane proteins with proteins on different cells (trans interactions) can drive the oligomerization of proteins on the same cell (cis interactions)2. A central problem in understanding the molecular basis of such phenomena is that equilibrium constants are generally measured in three-dimensional (3D) solution and are thus difficult to relate to the two-dimensional (2D) environment of a membrane surface. Here we present a theoretical treatment that converts 3D to 2D affinities accounting directly for the structure and dynamics of the membrane-bound molecules. Using a multi-scale simulation approach we apply the theory to explain the formation of ordered junction-like clusters by classical cadherin adhesion proteins. The approach includes atomic-scale molecular dynamics simulations to determine inter-domain flexibility, Monte-Carlo simulations of multi-domain motion, and lattice simulations of junction formation3. A finding of general relevance is that changes in inter-domain motion upon trans binding plays a crucial role in driving the lateral, cis, clustering of adhesion receptors.