Design of a photoswitchable cadherin

Abstract

There is a growing interest in engineering proteins whose function can be controlled with the spatial and temporal precision of light. Here, we present a novel example of a functional light-triggered switch in the Ca-dependent cell-cell adhesion protein E-cadherin, created using a mechanism-based design strategy. We report an 18-fold change in apparent Ca(2+) binding affinity upon illumination. Our results include a detailed examination of functional switching via linked changes in Ca(2+) binding and cadherin dimerization. This design opens avenues toward controllable tools that could be applied to many long-standing questions about cadherin's biological function in cell-cell adhesion and downstream signaling.

Figure 1

(A) A cartoon showing the basis of our design. As designed, our photoswitch reduces Ca²⁺ binding affinity, which, in turn, reduces homodimer affinity. (B) BSBCA undergoes a reversible cis/trans isomerization when illuminated with specific wavelengths of light. (C) EC12 structure showing the region targeted for photoswitchability. Labels indicate the design considerations. (D) Photoswitchability and reversibility measured by absorbance after many cycles of illumination of X-EC12.

Figure 2

Characterization of photoswitchable Ca²⁺ binding affinity. (A) Ca²⁺ binding as monitored by mass spectrometry. While WT and trans X-EC12 bound three Ca²⁺ ions specifically, cis X-EC12 showed considerably weaker, predominantly nonspecific binding (Figure S4 and SI). Fits are based on a model of a single class of binding site for a maximum of three specifically bound Ca²⁺ ions. (B) The half-life of the cis state as a function of Ca²⁺ concentration, as measured by absorbance. Error bars are +1 SD from three independent experiments.

Figure 3

Characterization of photoswitch homodimeric binding. (A) Homodimeric binding monitored in SPR as a function of Ca²⁺ concentration. The data were fit to a Hill equation. Faded points contain significant nonspecific binding and were not used in the fits. Responses between flow cells were scaled to minimize a least-squares difference, and then mean values were normalized such that the fit value at [Ca²⁺] = ∞ was 1.0 (SI). Error bars are ±1 SD of the three active flow cells in the instrument after scaling and normalization. Inset shows fits at low Ca²⁺ concentrations. (B) Homodimeric binding monitored in SPR at 1 mM Ca²⁺, after repeated illumination cycles. Responses between flow cells were scaled to minimize a least-squares difference. Error bars are ±1 SD of the three active flow cells in the instrument after scaling.