Abstract
Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.
Highlights
The most regulated cytoskeletal protein, filamentous actin (F-actin) is critical for cellular motility and mechanical strength, protein sorting and secretion, signal transduction, and cell division
Subsequent work (26) indicated that the chymotryptic GS-1 samples might been contaminated with a small amount of a longer fragment, containing segments-1–3 of gelsolin, which was responsible for the observed severing activity
Purification of GS-1-bound Actin Oligomers—We polymerized F-actin purified from chicken and treated it with pPDM, which cross-links between Cys-376 and Lys-193 of two adjacent actin protomers in a filament (17–19)
Summary
The most regulated cytoskeletal protein, filamentous actin (F-actin) is critical for cellular motility and mechanical strength, protein sorting and secretion, signal transduction, and cell division. The tendency of monomeric actin to form polymers of varying lengths has prevented crystallization and atomic resolution structural analysis of F-actin. All but one of the atomic resolution structures of actin are of the monomer bound to proteins that. The atomic coordinates and structure factors (code 1MDU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http:// www.rcsb.org/). A model describing F-actin as a helical polymer has been proposed based on fitting the crystal structure of monomeric actin into x-ray fiber diffraction data from oriented actin gels (1). Schutt and co-workers (4, 5, 10) have constructed a topologically different helical model for F-actin by twisting the ribbon-like assembly of actin molecules found in profilin-actin crystals. The ribbon actin assembly has been observed only in the crystals of profilin-actin heterodimers (4, 5, 10)
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