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. 2018 Jan 15;29(2):111-122.
doi: 10.1091/mbc.E17-03-0168. Epub 2017 Nov 15.

Salt bridges gate α-catenin activation at intercellular junctions

Samantha Barrick  1 , Jing Li  2   3   4 , Xinyu Kong  3 , Alokananda Ray  3 , Emad Tajkhorshid  2   3   4 , Deborah Leckband  5   2   3   4   6
Affiliations

Salt bridges gate α-catenin activation at intercellular junctions

Samantha Barrick et al. Mol Biol Cell. .

Abstract

Cadherin complexes transduce force fluctuations at junctions to activate signals that reinforce stressed intercellular contacts. α-Catenin is an identified force transducer within cadherin complexes that is autoinhibited under low tension. Increased force triggers a conformational change that exposes a cryptic site for the actin-binding protein vinculin. This study tested predictions that salt bridges within the force-sensing core modulate α-catenin activation. Studies with a fluorescence resonance energy transfer (FRET)-based α-catenin conformation sensor demonstrated that each of the salt-bridge mutations R551A and D503N enhances α-catenin activation in live cells, but R551A has a greater impact. Under dynamic force loading at reannealing cell-cell junctions, the R551A mutant bound more vinculin than wild-type α-catenin. In vitro binding measurements quantified the impact of the R551A mutation on the free-energy difference between the active and autoinhibited α-catenin conformers. A 2-μs constant-force, steered molecular dynamics simulation of the core force-sensing region suggested how the salt-bridge mutants alter the α-catenin conformation, and identified a novel load-bearing salt bridge. These results reveal key structural features that determine the force-transduction mechanism and the force sensitivity of this crucial nanomachine.

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Figures

FIGURE 1:
FIGURE 1:
α-Catenin structure. Structure from PDB ID 4IGG (Rangarajan and Izard, 2013). (A) α-Catenin consists of three major domains: the N-terminal (N, blue), modulatory (M), and C-terminal (C, red) domains. The M domain contains three four-helix bundles: MI (green/cyan), MII (yellow), and MIII (orange). MI contains the vinculin-binding site (cyan). The box indicates the region shown in B. (B) The salt bridge between D503 (MII domain) and R551 (MIII) is part of a salt-bridge network that stabilizes the structure of the M domain.
FIGURE 2:
FIGURE 2:
Apparent affinities between the vinculin head domain and WT or R551A α-catenin. The apparent dissociation constants between constitutively active vinculin (VHD, residues 1–252) and WT or R551A α-catenin measured using BLI. Traces on the left are the binding signal over time for different concentrations of VHD binding full-length α-catenin immobilized on a sensor by an N-terminal His6 tag. Graphs on the right show the steady-state analysis fits.
FIGURE 3:
FIGURE 3:
Vinculin recruitment at junctions between R2/7 cells expressing the WT or R551A α-catenin conformation sensor. Intensity ratios of immunostained vinculin and α-catenin at reannealing junctions between R2/7 cells cultured on collagen-coated glass substrates at intervals following a calcium switch. For the control condition, cells were not calcium switched but instead cultured on collagen-coated glass dishes overnight before fixation and immunostaining for vinculin and α-catenin. Intensity ratios represent the average of N = 3 independent experiments. Error bars represent SEM. * indicates p < 0.05.
FIGURE 4:
FIGURE 4:
Relative populations of unfurled α-catenin in cells expressing the WT, R551A, or D503N α-catenin conformation sensor. (A) Cartoon of cells expressing the α-catenin conformation sensor. Under tension, α-catenin undergoes a conformational change that separates the ECFP and YPet and reduces the FRET/ECFP ratio. (B) DIC (left) and FRET/ECFP (right) images for untreated or CytoD-treated MDCK cells transfected with WT, R551A, or D503N α-catenin sensor and cultured on collagen-coated glass. White boxes indicate ROIs at cell-cell junctions. Color scale indicates the absolute FRET/ECFP ratio. (C, D) FRET/ECFP ratios measured in cells expressing the WT, R551A, or D503N α-catenin conformation sensor. All ratios are normalized to the FRET/ECFP signal for untreated MDCK cells expressing the WT α-catenin conformation sensor. FRET/ECFP ratios at junctions and in the cytosol are normalized separately. For the +EGTA condition, FRET/ECFP ratios were measured after a 2-min treatment with 2 mM EGTA to disrupt cadherin-mediated junctions. For the +CytoD condition, cells were treated with 1 μM CytoD for 10 min before imaging. FRET/ECFP ratios represent the averages of N = 4 independent experiments for untreated cells and N = 2 independent experiments for EGTA- or CytoD-treated cells. Error bars represent SEM. * indicates p < 0.001. The numbers of analyzed cells and junctions for each condition are given in Supplemental Table S1. (C) FRET/ECFP in ROIs at junctions. (D) FRET/ECFP in cytosolic ROIs in MDCK cells. (E) Intensity ratios of α18 (activated) to anti–α-catenin (total) immunostained α-catenin at junctions between R2/7 cells cultured overnight on collagen-coated glass substrates. Intensity ratios are normalized to the α18/α-catenin ratio at junctions between cells expressing the WT conformation sensor. Intensity ratios represent the average of N = 2 independent experiments. Error bars represent SEM. * indicates p < 0.001.
FIGURE 5:
FIGURE 5:
Salt bridges at interdomain interfaces in the α-catenin M domain. Traces indicate the number of salt bridges between MIII and either MI or MII during the 2-μs constant-force SMD simulation of the α-catenin M domain.
FIGURE 6:
FIGURE 6:
Simulated unfolding of WT α-catenin under constant force. (A) Snapshots of α-catenin conformations at selected time points during a 2-μs SMD simulation of WT α-catenin M domain subject to a constant force of 100 pN. (See Supplemental Movie S1 for the entire trajectory.) Unraveled N-terminal helices are not shown for clarity. The light or dark gray box in the snapshot at 920 or 400 ns indicates a typical inset for the MI-MIII or MII-MIII interface, shown in B or C, respectively. (B, C) Insets showing salt bridges at interdomain interfaces at the simulation time points corresponding to the snapshots in A. (B) Salt bridges involving R326 at the MI-MIII interface of α-catenin. (C) Salt bridges involving R551 at the MII–MIII interface of α-catenin. (D, E) Evolution of distances between selected pairs of residues as a function of simulation time. Arrows indicate time points of snapshots in A–C. (D) D503-R551 is an interdomain salt bridge between MII and MIII; R326–D536 is a force-bearing contact between MI and MIII. (E) A316 and P635 are located near the ECFP and YPet of the α-catenin conformation sensor and are represented in A as blue and yellow spheres, respectively. G273 and P635 are the N- and C-terminal residues, respectively, of the M domain.
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