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Structural and thermodynamic characterization of cadherin·?-catenin·?-catenin complex formation.

ABSTRACT: The classical cadherin·?-catenin·?-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although ?-catenin binds to ?-catenin and to F-actin, ?-catenin significantly weakens the affinity of ?-catenin for F-actin. Moreover, ?-catenin self-associates into homodimers that block ?-catenin binding. We investigated quantitatively and structurally ?E- and ?N-catenin dimer formation, their interaction with ?-catenin and the cadherin·?-catenin complex, and the effect of the ?-catenin actin-binding domain on ?-catenin association. The two ?-catenin variants differ in their self-association properties: at physiological temperatures, ?E-catenin homodimerizes 10× more weakly than does ?N-catenin but is kinetically trapped in its oligomeric state. Both ?E- and ?N-catenin bind to ?-catenin with a Kd of 20 nM, and this affinity is increased by an order of magnitude when cadherin is bound to ?-catenin. We describe the crystal structure of a complex representing the full ?-catenin·?N-catenin interface. A three-dimensional model of the cadherin·?-catenin·?-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of ?-catenin has no influence on the interactions with ?-catenin, arguing against models in which ?-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of ?-catenin.

SUBMITTER: Pokutta S

PROVIDER: S-EPMC4036364 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Structural and thermodynamic characterization of cadherin·β-catenin·α-catenin complex formation.

Pokutta Sabine S Choi Hee-Jung HJ Ahlsen Goran G Hansen Scott D SD Weis William I WI

The Journal of biological chemistry 20140401 19

The classical cadherin·β-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to β-catenin and to F-actin, β-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block β-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with β-catenin and the cadherin·β ...[more]

PMID: 24692547

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Project description:DREAM (also known as K(+) channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca(2+)-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer's disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb(3+), are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb(3+). We also observe that unlike Tb(3+)-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (?average ? 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb(3+) binds to at least three sites with high affinity (Kd = 1.8 ?M in the presence of Ca(2+)) and displaces bound Ca(2+) through an entropically driven mechanism (?H ? 12 kcal mol(-1), and T?S ? 22 kcal mol(-1)). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb(3+), like Ca(2+), triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca(2+) binding, Tb(3+) binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry-mass spectrometry reveal that replacement of Ca(2+) with Tb(3+) preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM-protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.

Dataset Information

Structural and thermodynamic characterization of cadherin·?-catenin·?-catenin complex formation.

Publications

Structural and thermodynamic characterization of cadherin·β-catenin·α-catenin complex formation.

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