Allosteric cross talk between cadherin extracellular domains

Abstract

Atomic force microscopy and surface force apparatus measurements determined the functional impact of the cadherin point mutation W2A and domain deletion mutations on C-cadherin binding signatures. Direct comparison of results obtained using both experimental approaches demonstrates that C-cadherin ectodomains form multiple independent bonds that require different structural regions. The results presented reveal significant interdomain cross talk. They further demonstrate that the mutation W2A not only abolishes adhesion between N-terminal domains, but allosterically modulates other binding states that require functional domains distal to the N-terminal binding site. Such allosteric effects may play a prominent role in modulating adhesion by Type I classic cadherins, cadherin oligomerization at junctional contacts, and propagation of binding information to the cytoplasmic region.

Figure 1

(A) Schematic of sample configurations used in SFA measurements. Cadherins are immobilized on supported lipid bilayers. D, interbilayer distance; T, distance between DPPE monolayers. Here, CEC1–5-His₆ is immobilized on the upper membrane and W2A-His₆ on the lower membrane. (B) Schematic of sample configuration in AFM measurements. The proteins are covalently bound to SAMs on the tip and glass slide via 3400-mol wt PEG linkers. (C) Typical force-extension curves measured with the AFM at r_F = 400 nm/s, showing contact (arrow) followed by either no binding (top) or formation of a single bond (middle trace) and multiple bonds (bottom) upon tip retraction. The solid line in the middle trace of C shows the linear fit to the force-extension curve just before bond rupture (asterisk). The slope of this line is used to determine the pulling rate just before bond rupture.

Figure 2

Normalized force-distance profiles between oriented monolayers of CEC1–5-His₆ and W2A-His₆. Solid circles indicate forces measured during approach, and open circles indicate forces measured during separation. (A) Normalized force-distance curves between oriented monolayers of CEC1–5-His₆. Right-pointing arrows indicate bond failure and jumps out of contact. Error bars represent the mean± SD. Here, the inner bond is at 252 ± 6 Å (i), the middle bond at 314 ± 6 Å (m), and the outer bond at 402 ± 6 Å (o). Vertical dashed lines show the alignment of the three adhesive minima in the different measurements. (B) Normalized force-distance curves between oriented W2A-His₆ monolayers. Left-pointing arrows indicate jumps into contact. (C) Normalized force-distance curves measured between W2A and CEC1–5-His₆.

Figure 3

Estimated adhesion energies (in k_BT) for the inner, middle, and outer bonds for the cadherin interactions CEC1–5/CEC1–5, W2A/W2A, and CEC1–5/W2A. The bars are the means and error bars indicate the mean ± SD. ^∗∗p < 0.01 for the difference in adhesion energies between the inner and middle bonds of the W2A/W2A and CEC1–5/ CEC1–5 bonds.

Figure 4

(A–E) Representative rupture force distributions between CEC12-Fc (A), CEC1–5-Fc (B), CEC1–3 (C), and CEC1–4 (D) fragments at the indicated pulling rates. (E) The most probable force, F_mp, of each bound state plotted against the logarithm of the pulling rate r_F. The solid lines are linear fits to the force spectra with best-fit parameters summarized in Table 2. Error bars smaller than the size of the symbols are not shown. Solid lines in A–D are probability distributions computed using the parameters obtained from the force spectra in E.

Figure 5

Models of cadherin binding consistent with the data. (A) Cis (lateral dimers) stabilized by an EC3-dependent bond (gray domain). (B) The formation of trans (adhesive) dimers between EC1 domains on opposing proteins could lead to the accumulation of cadherins locally to promote the formation of cis dimers through EC3-dependent bonds (gray domains). (C) In force measurements, and possibly on the cell surface, cis bonds (gray) can also form between cadherins on opposing probe surfaces. The force measurements would sample adhesive states due to both lateral and adhesive dimers. (D) Proteins form antiparallel adhesive bonds stabilized by interactions between EC3 domains.