Structure of an Actin Spring

Scruin, a member of the ß-propeller superfamily, crosslinks actin filaments into a crystalline bundle (acrosomal process) in the Limulus sperm. It consists of a ~103 kD heavy chain and a ~17 kD calmodulin light chain. 3D reconstruction of acrosomal process show that scruin wraps around the outside of the actin filament and that scruin consists of 2 domains which contact the underlying actin filament and make crosslinks to scruin molecules on neighboring filaments. A model shows an actin filament decorated by bi-lobed scruin molecules. One domain of scruin is bound to one actin subunit while the other domain is bound to a neighboring subunit. A 3D reconstruction of the bundle has been calculated from exhaustive EM tilt series and solution x-ray scattering. In the bundle, a single filament appears to contact two adjacent filaments through the outer scruin molecules. Surprisingly, a single scruin molecule may bind to three scruin molecules on the neighboring two filaments. This finding suggests a more complex mechanism of crosslinking that relies on multiple scruin-scruin interactions. Furthermore, the change in interactions during the acrosome reaction may involve modulation of the scruin conformation. EM and analytical ultracentrifugation studies document a calcium-dependent conformation that involves a relaxation of the scruin structure. [P.Matsudaira, G.Waller in collaboration with M.Sherman, M.Schmid, and W. Chiu, Baylor College of Medicine]

Crystallographic reconstruction of the acrosomal bundle showing extensive interfilament interactions (yellow). These interactions are correlated with the scruin domains. Interactions involving spheroidal scruin domains of the central filament are shown by red diamonds whereas those involving elongated domains are shown by red asterisks.

Unleashing an Actin Spring

Poorly understood aspects of actin dynamics include the movements and forces generated when a bundle of actin filaments twists or bends. The best example of torsion-generating force and large movements is the uncoiling of the acrosomal bundle into a 50 µm- long acrosomal process. Supported by the bundle of filaments, a finger of membrane bridges between sperm and egg to initiate fertilization of the horseshoe crab, Limulus polyphemus. Our lab studies the structure of the actin bundle, the energetics of converting the potential energy of a macromolecular spring into the extension of a crystalline bundle, and the mechanism by which the crosslinking protein, scruin, controls these changes in actin structure. [P.Matsudaira, G.Waller in collaboration with J.Shin and L.Mahadevan, MIT Department of Mechanical Engineering]

In its native state, the acrosomal bundle consists of a 60µm para-crystalline helical coil of bent, twisted actin filaments. In the presence of calcium, the actin binding protein, scruin, undergoes a conformation change, which causes the individual actin filaments to untwist. This leads to the straightening of the 60µm long bundle which is propelled through a nuclear channel at a mean velocity of 15µm/s at room temperature (24-26°C). Its velocity is constant throughout the entire extension, suggesting that the uncoiling of the bundle is a localized event that propagates in a zipper-like fashion. The average velocity of the acrosomal process depends on the temperature and increases as the temperature is raised, varying from approximately 37µm/s at 32°C to 1.7µm/s at 9.6°C. Based on dynamical measurements of the uncoiling and the extension of the actin bundle, we estimated the energy dissipated hydrodynamically during the extension to be of the order of 10^-8 ergs. The bending stiffness, EI, of the actin bundle was measured by analyzing the bending shape at equilibrium in a steady hydrodynamic flow and was of the order of 10^-20 Nm² leading to an estimate for the initially stored energy of 7•10^-6 ergs. Therefore, the acrosomal bundle indeed behaves like a mechanical spring, and the strain energy is the major source of the energy that powers the acrosomal reaction to completion.