Actin

Actin filaments (F-actin) are found in most eukaryotic cells as constituents of the cytoskeleton. They play a central role in various types of motility (including muscle contraction) and transport processes. A major step toward understanding how actin can fulfill its various functions was the determination of the atomic structure of monomeric actin (G-actin) complexed to accessory proteins (Kabsch et al., 1990). Combining the atomic structure of G-actin with fiber diffraction patterns obtained from oriented gels of actin filaments enabled Holmes and co-workers to construct an atomic model of the F-actin filament (i.e., the Holmes-Lorenz model; Holmes et al., 1990; Lorenz et al., 1993). This filament model has provided a structural framework for a still growing number of biochemical, cell biological and mechanical investigations on actin, including mapping of the binding sites of various accessory proteins and drugs at atomic scale. More recently, Schutt and colleagues have constructed an alternative atomic model of F-actin which they derived from the structural analysis of bovine profilin-b-actin co-crystals (i.e., the Schutt-Lindberg model; Schutt et al., 1997). Their ribbon-based filament model is substantially different from the Holmes-Lorenz model, although it is corroborated by the same structural constraints as were used to build and refine the former. Moreover, Schutt and co-workers have used their F-actin model as a key element in a recent hypothesis of the mechanism of muscle contraction (Schutt et al., 1997). Hence, it is necessary to more critically evaluate the two models and to arrive at a consensus structure of the F-actin filament at atomic scale.

Towards a consensus structure of the F-actin filament at atomic scale, we have prepared an undecagold-tagged phalloidin derivative (Au11-phalloidin) to determine this mushroom toxin's binding site and orientation within the F-actin filament by scanning transmission electron microscopy (STEM) and 3-D helical reconstruction (Fig. 1). The structural data obtained when combined with various biochemical constraints enabled us to critically evaluate two distinct atomic models of the Factin filament (i.e., the Holmes-Lorenz versus the Schutt-Lindberg model). Taken together, our data are in excellent agreement with the Holmes-Lorenz model.

Figure 1: Structural analysis of native, phalloidin (PHD) stabilized, and Au11-PHD stabilized rabbit muscle F-actin filaments and orientation of PHD within its F-actin binding site. (a) STEM dark-field micrograph of a negatively stained PHD stabilized F-actin filament stretch. (b) As (a), but freeze-dried and unstained.

(c) Unstained and freeze-dried Au11-PHD stabilized F-actin filament stretch. (d) As (c) but contrast adjusted so as to display the highest intensities only, which correspond to single Au11-clusters (diameter ~1 nm) spaced approximately every 5.5 nm along the two long-pitch helical strands which are staggered by 2.75 nm relative to one another (see yellow lines). (e) An averaged and refined 3-D helical reconstruction computed from negatively stained Au11-PHD stabilized F-actin filament stretches (see a) has been surface-rendered to include 100% of its nominal molecular mass. The location of the Au11-clusters has been determined from a difference map (i.e., Au11-PHD stabilized F-actin filament reconstruction minus PHD stabilized F-actin filament reconstruction) and visualized by 1-nm diameter gold spheres. (f) Alignment and overlay of an atomic PHD stabilized F-actin trimer (yellow ribbon with the Au11-PHD:F-actin 3-D reconstruction). Scale bar, 25 nm (a-d).

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