Sanchaita Das1, Peng Ge2, Zeynep A Oztug Durer1, Elena E Grintsevich1, Z Hong Zhou3, Emil Reisler4. 1. Department of Chemistry and Biochemistry, University of California, UCLA, Los Angeles, CA 90095, USA. 2. California NanoSystems Institute (CNSI), UCLA, Los Angeles, CA 90095, USA. 3. California NanoSystems Institute (CNSI), UCLA, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, CA 90095, USA; Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA. 4. Department of Chemistry and Biochemistry, University of California, UCLA, Los Angeles, CA 90095, USA; Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA. Electronic address: reisler@mbi.ucla.edu.
Abstract
Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.
Detailed molecular information on G-actin assembly into filaments (F-n class="Gene">actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeastactin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.
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