The cadherin–catenin adhesion complex is the central component of the cell–cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•β-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and β-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of β-catenin forms a flexible “tongue” that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin–catenin adhesion complex in mechanotransduction.
more »
« less
Activated nanoscale actin-binding domain motion in the catenin–cadherin complex revealed by neutron spin echo spectroscopy
As the core component of the adherens junction in cell–cell adhesion, the cadherin–catenin complex transduces mechanical tension between neighboring cells. Structural studies have shown that the cadherin–catenin complex exists as an ensemble of flexible conformations, with the actin-binding domain (ABD) of α-catenin adopting a variety of configurations. Here, we have determined the nanoscale protein domain dynamics of the cadherin–catenin complex using neutron spin echo spectroscopy (NSE), selective deuteration, and theoretical physics analyses. NSE reveals that, in the cadherin–catenin complex, the motion of the entire ABD becomes activated on nanosecond to submicrosecond timescales. By contrast, in the α-catenin homodimer, only the smaller disordered C-terminal tail of ABD is moving. Molecular dynamics (MD) simulations also show increased mobility of ABD in the cadherin–catenin complex, compared to the α-catenin homodimer. Biased MD simulations further reveal that the applied external forces promote the transition of ABD in the cadherin–catenin complex from an ensemble of diverse conformational states to specific states that resemble the actin-bound structure. The activated motion and an ensemble of flexible configurations of the mechanosensory ABD suggest the formation of an entropic trap in the cadherin–catenin complex, serving as negative allosteric regulation that impedes the complex from binding to actin under zero force. Mechanical tension facilitates the reduction in dynamics and narrows the conformational ensemble of ABD to specific configurations that are well suited to bind F-actin. Our results provide a protein dynamics and entropic explanation for the observed force-sensitive binding behavior of a mechanosensitive protein complex.
more » « less- Award ID(s):
- 1817684
- NSF-PAR ID:
- 10218373
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 13
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- Article No. e2025012118
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Attachment between cells is crucial for almost all aspects of the life of cells. These inter-cell adhesions are mediated by the binding of transmembrane cadherin receptors of one cell to cadherins of a neighboring cell. Inside the cell, cadherin binds β-catenin, which interacts with α-catenin. The transitioning of cells between migration and adhesion is modulated by α-catenin, which links cell junctions and the plasma membrane to the actin cytoskeleton. At cell junctions, a single β-catenin/α-catenin heterodimer slips along filamentous actin in the direction of cytoskeletal tension which unfolds clustered heterodimers to form catch bonds with F-actin. Outside cell junctions, α-catenin dimerizes and links the plasma membrane to F-actin. Under cytoskeletal tension, α-catenin unfolds and forms an asymmetric catch bond with F-actin. To understand the mechanism of this important α-catenin function, we determined the 2.7 Å cryogenic electron microscopy (cryoEM) structures of filamentous actin alone and bound to human dimeric α-catenin. Our structures provide mechanistic insights into the role of the α-catenin interdomain interactions in directing α-catenin function and suggest a bivalent mechanism. Further, our cryoEM structure of human monomeric α-catenin provides mechanistic insights into α-catenin autoinhibition. Collectively, our structures capture the initial α-catenin interaction with F-actin before the sensing of force, which is a crucial event in cell adhesion and human disease.more » « less
-
more » « less
The cytoplasmic tails of classical cadherins form a multiprotein cadherin–catenin complex (CCC) that constitutes the major structural unit of adherens junctions (AJs). The CCC in AJs forms junctional clusters, “E clusters,” driven by
cis andtrans interactions in the cadherin ectodomain and stabilized by α-catenin–actin interactions. Additional proteins are known to bind to the cytoplasmic region of the CCC. Here, we analyze how these CCC-associated proteins (CAPs) integrate into cadherin clusters and how they affect the clustering process. Using a cross-linking approach coupled with mass spectrometry, we found that the majority of CAPs, including the force-sensing protein vinculin, interact with CCCs outside of AJs. Accordingly, structural modeling shows that there is not enough space for CAPs the size of vinculin to integrate into E clusters. Using two CAPs, scribble and erbin, as examples, we provide evidence that these proteins form separate clusters, which we term “C clusters.” As proof of principle, we show, by using cadherin ectodomain monoclonal antibodies (mAbs), that mAb-bound E-cadherin forms separate clusters that undergotrans interactions. Taken together, our data suggest that, in addition to its role in cell–cell adhesion, CAP-driven CCC clustering serves to organize cytoplasmic proteins into distinct domains that may synchronize signaling networks of neighboring cells within tissues. -
Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca 2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca 2+ -induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin–actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca 2+ -induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca 2+ -troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length–dependent enhancement of troponin regulation with both Ca 2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length–dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length–dependent Ca 2+ -troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca 2+ -troponin regulation of the myocardium.more » « less
-
E-cadherin plays a central role in cell-cell adhesion. The ectodomains of wild type cadherins form a crystalline- like two dimensional lattice in cell-cell interfaces mediated by both trans (apposed cell) and cis (same cell) interactions. In addition to these extracellular forces, adhesive strength is further regulated by cytosolic phenomena involving 𝛼 and 𝛽- catenin–mediated interactions between cadherin and the actin cytoskeleton. Cell-cell adhesion can be further strengthened under tension through mechanisms that have not been definitively characterized in molecular detail. Here we quantitatively determine the role of the cadherin ectodomain in mechanosensing. To this end, we devise an E-cadherin-coated emulsion system, in which droplet surface tension is balanced by protein binding strength to give rise to stable areas of adhesion. To reach the honeycomb/cohesive limit, an initial emulsion compression by centrifugation facilitates E-cadherin trans-binding, while a high protein surface concentration enables the cis-enhanced stabilization of the interface. We observe an abrupt concentration dependence on recruitment into adhesions of constant crystalline density, reminiscent of a first-order phase transition. Removing the lateral cis-interaction with a "cis mutant" shifts this transition to higher surface densities leading to denser, yet weaker adhesions. In both proteins, the stabilization of progressively larger areas of deformation can be rationalized by a stiffening catch-bond, whose strength increases with tension. This catch bond may well correspond to one that has been identified in the cadherin “X-dimer".more » « less
