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Binding partner- and force-promoted changes in ?E-catenin conformation probed by native cysteine labeling.

ABSTRACT: Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via ?-catenin and the F-actin binding protein ?E-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through ?E-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in ?E-catenin upon binding to the E-cadherin•?-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in ?E-catenin when bound to F-actin. Comparisons of wild-type ?E-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal ?-catenin-binding and the middle (M) vinculin-binding domain of ?E-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length ?E-catenin, and identify the M domain as a key transmitter of conformational changes.

SUBMITTER: Terekhova K

PROVIDER: S-EPMC6814714 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Binding partner- and force-promoted changes in αE-catenin conformation probed by native cysteine labeling.

Terekhova Ksenia K Pokutta Sabine S Kee Yee S YS Li Jing J Tajkhorshid Emad E Fuller Gerald G Dunn Alexander R AR Weis William I WI

Scientific reports 20191025 1

Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin up ...[more]

PMID: 31653927

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Project description:Microsecond-long all-atom molecular dynamics (MD) simulations, circular dichroism, laser Doppler velocimetry, and dynamic light-scattering techniques have been used to investigate pH-induced changes in the secondary structure, charge, and conformation of poly l-lysine (PLL) and poly l-glutamic acid (PGA). The employed combination of the experimental methods reveals for both PLL and PGA a narrow pH range at which they are charged enough to form stable colloidal suspensions, maintaining their α-helix content above 60%; an elevated charge state of the peptides required for colloidal stability promotes the peptide solvation as a random coil. To obtain a more microscopic view on the conformations and to verify the modeling performance, peptide secondary structure and conformations rising in MD simulations are also examined using three different force fields, i.e., OPLS-AA, CHARMM27, and AMBER99SB*-ILDNP. Ramachandran plots reveal that in the examined setup the α-helix content is systematically overestimated in CHARMM27, while OPLS-AA overestimates the β-sheet fraction at lower ionization degrees. At high ionization degrees, the OPLS-AA force-field-predicted secondary structure fractions match the experimentally measured distribution most closely. However, the pH-induced changes in PLL and PGA secondary structure are reasonably captured only by the AMBER99SB*-ILDNP force field, with the exception of the fully charged PGA in which the α-helix content is overestimated. The comparison to simulations results shows that the examined force fields involve significant deviations in their predictions for charged homopolypeptides. The detailed mapping of secondary structure dependency on pH for the polypeptides, especially finding the stable colloidal α-helical regime for both examined peptides, has significant potential for practical applications of the charged homopolypeptides. The findings raise attention especially to the pH fine tuning as an underappreciated control factor in surface modification and self-assembly.

Dataset Information

Binding partner- and force-promoted changes in ?E-catenin conformation probed by native cysteine labeling.

Publications

Binding partner- and force-promoted changes in αE-catenin conformation probed by native cysteine labeling.

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