Atomic Conformational Dynamics and Actin-Crosslinking Function of Alpha-Actinin Revealed by SimHS-AFMfit

Alpha-actinin is a crucial actin-binding protein involved in cellular functions like muscle contractility, mechanosensation, and cell division. Studying its atomic structure and dynamics is challenging due to protein complexity and imaging limitations. To address this, we developed SimHS-AFMfit , integrating high-speed AFM (HS-AFM), AFMfit (a flexible fitting technique), and molecular dynamics (MD) simulations. This approach successfully converted 2D AFM images into 3D atomic conformations, revealing the atomic conformational dynamics of Ca²⁺-bound and unbound alpha-actinin. Using principal component analysis, we classified and quantified protein motions such as twisting and bending. HS-AFM and MD simulations further characterized the fluctuations of rise and axial distance (AD) between adjacent protomers in actin filament crosslinking models, identifying structural features that regulate myosin II and cofilin binding. These findings enhance our understanding of alpha-actinin’s role in actin filament modulation and provide insights for studying more complex protein assemblies, such as contractile rings.