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Constantina Bakolitsa

Publications and source records attributed to Constantina Bakolitsa.

4 recordsLinked to original sources
PubMed2006

Structure of the alpha-actinin-vinculin head domain complex determined by cryo-electron microscopy.

The vinculin binding site on alpha-actinin was determined by cryo-electron microscopy of 2D arrays formed on phospholipid monolayers doped with a nickel chelating lipid. Chicken smooth muscle alpha-actinin was cocrystallized with the beta1-integrin cytoplasmic domain and a vinculin fragment containing residues 1-258 (vinculin(D1)). Vinculin(D1) was located at a single site on alpha-actinin with 60-70% occupancy. In these arrays, alpha-actinin lacks molecular 2-fold symmetry and the two ends of the molecule, which contain the calmodulin-like and actin binding domains, are held in distinctly different environments. The vinculin(D1) difference density has a shape very suggestive of the atomic structure. The atomic model of the complex juxtaposes the alpha-actinin binding site on vinculin(D1) with the N-terminal lobe of the calmodulin-like domain on alpha-actinin. The results show that the interaction between two species with weak affinity can be visualized in a membrane-like environment.

Actinin↗
PubMed2005

Two-wavelength MAD phasing and radiation damage: a case study.

Radiation damage affects MAD experiments in two ways: (i) increased absorption by the crystal at the wavelengths of interest for the experiment results in faster crystal deterioration; (ii) lack of isomorphism induced by radiation damage causes problems when scaling and merging data at different wavelengths and can prevent accurate measurement of anomalous and dispersive differences. In an attempt to overcome these problems in the case of radiation-sensitive crystals of vinculin, two-wavelength MAD data were collected at the Se absorption-edge inflection and at high-energy remote wavelengths. Although this strategy resulted in a lower total absorbed dose compared with a standard three-wavelength experiment using the peak wavelength, an increase in the unit-cell volume and other effects attributable to radiation damage were still observed. In an effort to extract the maximum information available from the data, different data-processing and scaling procedures were compared. Scaling approaches involving local scaling of unmerged reflections were consistently successful and most ordered Se sites could be located. Subsequent use of these sites for phasing resulted in an interpretable electron density map. This case demonstrates the feasibility of two-wavelength MAD in the presence of moderate radiation damage using conventional data collection strategies and widely available standard software.

Crystallization↗
PubMed2004

Structural basis for vinculin activation at sites of cell adhesion.

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.

Allosteric Regulation↗