Actin crystal dynamics: structural implications for F-actin nucleation, polymerization, and branching mediated by the anti-parallel dimer Robbie Reutzel, a,1 Craig Yoshioka, a,1,2 Lakshmanan Govindasamy, a Elena G. Yarmola, b Mavis Agbandje-McKenna, a Michael R. Bubb, a,b and Robert McKenna a, * a Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610, USA b The Research Service, Malcolm Randall Department of Veterans Affairs Medical Center and University of Florida College of Medicine, Gainesville, FL 32608, USA Received 9 September 2003, and in revised form 10 December 2003 Abstract Actin filament nucleation, polymerization, and branching are crucial steps in many forms of cell motility, cell shape, and in- tracellular organelle movements in a wide range of organisms. Previous biochemical data suggests that an anti-parallel actin dimer can incorporate itself into growing filamentous actin (F-actin) and has a role in branching. Furthermore, it is a widespread belief that nucleation is spawned from an actin trimer complex. Here we present the structures of actin dimers and trimers in two te- tragonal crystal systems P4 3 2 1 2 and P4 3 . Both crystal systems formed by an induced condensation transformation of a previously reported orthorhombic crystal system P2 1 2 1 2 1 . Comparison between the three crystal systems demonstrates the dynamics and flexibility of actin–actin interactions. The dimer and trimer actin rearrangements observed between the three crystal systems may provide insight to in vivo actin–actin interactions that occur during the nucleation, polymerization, and branching of F-actin. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Actin; Polymerization; Nucleation; Branching; Dynamics 1. Introduction Actin is a ubiquitously expressed protein of 375 amino acids encoded by a large, highly conserved gene family. Some single cell organisms like yeast have a single actin gene whereas humans have six actin genes and some plants have as many as 60 (Hennessey et al., 1993). Sequence comparisons of actin from amoebas and vertebrates display remarkable conservation; shar- ing 80% sequence identity (Gallwitz and Sures, 1980). Actin is the most abundant cytosolic protein in eu- karyotic cells, making up 10% weight of the total cell protein in muscle and 1–5% of the total weight in non- muscle cells. Actin exists in the cell as a globular monomer termed G-actin and as a filamentous, helical polymer named F-actin. Each actin monomer contains a divalent (Mg 2þ or Ca 2þ ) ion complexed with either ATP or ADP (De La Cruz and Pollard, 1995; De La Cruz et al., 2000; Steinmetz et al., 1997). The nucleation, elongation, and branching of actin filaments have been shown to be the driving force in many forms of patho- genic cell motility as well as pinocytosis and organelle movement (Ploubidou and Way, 2001; Taunton, 2001). The rapidity with which these constructive events occur emphasizes the efficiency and dynamic nature of both the nucleation mechanism and the actin monomer itself. The addition of millimolar concentrations of salts to G-actin solutions induces F-actin polymerization. The investigation of F-actin formation and assembly, with and without different divalent cations, has revealed variation in polymerization speed and assembly dy- namics (Steinmetz et al., 1997). Such an array of F-actin * Corresponding author. Fax: 1-352-392-3422. E-mail address: rmckenna@ufl.edu (R. McKenna). 1 These authors contributed equally to this work. 2 Present address: The Scripps Research Institute, La Jolla, CA 92037, USA. 1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.12.006 Journal of Structural Biology 146 (2004) 291–301 Journal of Structural Biology www.elsevier.com/locate/yjsbi