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|Title:||Crystal structure of the vinculin tail domain and a model for activation|
|Presented at:||University of Leicester|
|Abstract:||Basic physical mechanisms underlying the regulation of focal adhesion formation and function include aggregation, phosphorylation and conformational changes of the molecules involved.;Vinculin is a protein located at sites of cell-cell and cell-matrix adhesion where it plays a dynamic role in the assembly of the actin cytoskeleton. A strong interaction between its head and tail domains that regulates binding to other cytoskeletal components is disrupted by acidic phospholipids. The crystal structure of the vinculin tail, residues 789-1066, was determined by the method of single isomorphous replacement with anomalous scattering. Five amphipathic helices form an antiparallel bundle that resembles exchangeable apolipoproteins. A C-terminal arm wraps across the base of the bundle and emerges as a hydrophobic hairpin surrounded by a collar of basic residues, adjacent to the N-terminus.;A series of structure-designed solution studies were carried out on the vinculin tail and a C-terminal deletion mutant. Co-sedimentation assays showed that the C-terminal arm is required for binding to acidic phospholipids but not to F-actin, while limited proteolysis and stopped-flow measurements indicated that binding to either ligand induces conformational changes in the N- and C-terminal regions of the molecule. These changes may represent the first step in a model for the activation of the intact vinculin, where PIP2-triggered unfurling of the tail domain would lead to subsequent sequestration of the head and neck regions allowing binding to F-actin.;Crystals of the intact vinculin diffracting synchrotron radiation to medium resolution have also been obtained.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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