The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca2+-dependent actin binding

The three-dimensional structure of alpha -actinin from rabbit skeletal musc le was determined by cryoelectron microscopy in combination with homology m odeling of the separate domain structures based on results previously deter mined by X-ray crystallography and nuclear magnetic resonance spectroscopy, alpha -Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, froz en hydrated specimens at tilt angles from 0 degrees to 60 degrees. Interpre tation of the 15 Angstrom -resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-bindi ng, three-helix motif and the C-terminal calmodulinlike domains. The initia l model was refined quantitatively to improve its fit to the experimental r econstruction. The molecular model of alpha -actinin provides the first vie w of the overall structure of a complete actin crosslinking protein. The st ructure is characterized by close proximity of the C-terminal, calmodulin-l ike domain to the linker between the two calponin-homology domains that com prise the actin-binding domain. This location suggests a hypothesis to expl ain the involvement of the C-terminal domain in Ca2+-dependent actin bindin g of non-muscle isoforms. (C) 2001 Academic Press.