Structure of an Actin Trimer Stabilized by a Tandem W Domain Hybrid Construct

Abstract

The study of the actin filament is one of the major problems of structural biology. Uncontrollable polymerization has interfered with our ability to obtain crystals of the filament. Proteins that initiate actin polymerization in cells have the natural ability to stabilize multiple actin subunits into a filament-like conformation, allowing them to overcome the rate-limiting step in polymerization, i.e. the formation of actin dimers and trimers. With the exception of formins, known filament nucleators use the Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. The W domain is a short (17–27 aa) actin-binding motif. A common architecture, found in filament nucleators such as Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. We have engineered a stable actin trimer stabilized by a tandem W domain hybrid construct that also includes filament barbed and pointed end capping elements. The structure of the actin trimer was first studied in solution using x-ray scattering. Different crystal forms of the trimer have been obtained. We will present these studies. In particular, we will show how tandem W domains stabilize a polymerization nucleus by organizing actin subunits into a filament-like conformation.Supported by NIH Grant HL086655