This project studies the molecular basis for actin cytoskeleton remodeling in vascular smooth musclecontraction. It complements the other projects of this grants that focus on tissues, cells and macromolecularassemblies. Two families of actin-binding proteins, Ena/VASP and WASP, have emerged as key regulatorsof cytoskeleton remodeling, playing distinct roles in filament nucleation and elongation, respectively. Themolecular mechanisms controlling both processes remain a mystery. Ena/VASP and WASP are functionallydistinct but share similar modular structures. They both contain poly-Pro regions that mediate the binding ofprofilin-actin, followed by G-actin binding domains of the WASP-Homology 2 (WH2) type. This project buildsupon important preliminary results (presented here) and our recently determined structures of various WH2-actin complexes to propose the following two hypotheses: 1) Tandem WH2s line up actin subunits along afilament strand forming nuclei for actin assembly (nucleation step). 2) The poly-Pro-WH2 module contributesto filament elongation by 'processing' profilin-actin complexes for their incorporation onto the barbed end ofgrowing filaments (elongation step). To test these hypotheses, aims 1 and 2 will dissect the structure-function of the poly-Pro-WH2 and tandem-WH2 modules. We will determine the crystal structures of actinminifilaments assembled via tandem WH2 hybrid constructs and that of poly-Pro-WH2 bound to profilin-actin(transition state in elongation). Complementary solution studies will be carried out using analyticalultracentrifugation and SAXS/WAXS. A biophysical study of the various protein-protein interactions involvedin nucleation and elongation will investigate the role of allosteric effects in these processes.
Aim 3 studiesthe actin binding and scaffolding functions of alpha-actinin, a key adaptor protein of the spectrin family.Alpha-actinin mediates the interactions between integrins at the plasma membrane and the focal-adhesionproteins zyxin, vinculin, and paladin, which in turn recruit Ena/VASP to dense plaques. As a paradigm ofthese interactions, we will study the structural basis for the alpha-actinin-zyxin interaction. UsingSAXS/WAXS, FRET and AU, we will test the two prevailing F-actin-binding models, compact and extended,for the actin-binding domain of members of the spectrin family. Understanding the molecular basis ofvascular muscle contraction will accelerate the discovery of therapies to treat cardiovascular diseases.
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