Structural aspects of actin-binding proteins Michael D Rozycki, James C Myslik, Clarence E Schutt and Uno Lindberg Princeton University, Princeton, USA and Stockholm University, Stockholm, Sweden The three-dimensional structures of myosin subfragment 1 (5~11, gelsolin segment 1 complexed with a-actin, villin fragment 14T, Acanthamoeba profilin-I, and bovine profilin complexed with p-actin were completed last year. Together, they expand our understanding of the structural organization of actin-binding proteins. In addition, the segment 1 and bovine profilin complexes provide atomic-level descriptions of their interfaces with actin. Current Opinion in Cell Biology 1994, 6337-95 Introduction The crystal structure of monomeric a-actin complexed with DNase I 11) enabled cell biologists to begin to un- derstand actin-binding proteins in terms of specific interactions with actin. A model for the actin Iila- merit based on the monomer structure 121 provided a starting point for discussing the mechanisms by which actin-binding proteins combine with F-actin to carry out functional reorganizations of the cytoskele- ton. Last year, the three-dimensional structures of sev- eral actin-binding proteins were completed, including those of myosin subfragment 1, gelsolin segment 1 complexed with a-actin, villin fragment 14T, Acan- thamoeba profilin-I, and bovine profilin complexed with l!l-actin. The gelsolin segment 1 and bovine pro- filin structures provide atomic resolution views of their respective interfaces with actin and, along with the structure of the DNase I : a-actin complex, reveal sites on actin that are responsible for binding accessory pro- teins. This review will focus on structural aspects of actin-binding proteins that relate to their interactions with actin. Myosin Sl Intact skeletal muscle myosin contains two identical headpieces, denoted subfragment 1 (Sl), that can be removed by papain hydrolysis. Sl, composed of a 95 kDa heavy chain and two light chains, has a total molecular mass of 130 kDa [31 and possesses binding sites for nucleotide and actin [4]. The three-dimensional structure of chicken pectoralis Sl was recently solved to 2.8 A resolution using multiple isomorphous replace- ment phasing P.1. Crystallization depended on reduc- tive methylation of almost all of its 106 lysine residues, either to reduce solubility or to stabilize the conforma- tion. White and Rayment WI were careful to show that such extensive modification did not inactivate the Sl ATPase, and Rypniewski et al. [7*1 showed that similar methylation of hen egg lysozyme did not significantly altef the conformation of the protein at a resolution of 1.8A, except at surface loops. The structure of myosin Sl l5”l is shown in Figure 1. The predominant element is the globular head, composed of the heavy chain. This heavy chain is divided into three fragments by tryptic digestion: a 25 kDa fragment at the amino terminus containing the nucleotide-binding site, a 50 kDa fragment in the mid- dle of the sequence, which is divided into an upper and a lower domain divided by a cleft [5**1, and a 20 kDa fragment at the carboxyl terminus. The latter two frag- ments share the actin-binding site. Extending from the globular head is a helix 85A in length at the carboxyl terminus of the heavy chain. The regulatory light chain wraps around this helix in such a way that the carboxyl termini of the two chains are in close proximity. This chain shows structural homology with calmodulin and troponin C, and contains a calcium-binding site. The essential light chain also wraps around the long helix, fitting between the regulatory light chain and the glob- ular head of the heavy chain adjacent to the nucleotide- binding region. This chain is the most disordered part of the Sl structure, but it is not clear whether this is due to conformational flexibility of the protein or to crystal non-isomorphism [5”1. Although Sl was crystallized in the absence of ATP, the nucleotide-binding site was identified by the resem- blance of a phosphate-binding loop in the 25 kDa heavy chain fragment to those found in Ras protein and adenylate cyclase [5”1. The nucleotide pocket is approximately 15A in diameter, and includes Cys697 and Cys707. These residues can be crosslinked un- der a variety of conditions, but only in the presence of nucleotide, suggesting that nucleotide binding in- duces a significant conformation change in the pocket PI. The actin-binding domain in myosin lies on the face opposite the nucleotide-binding site. The elec- Abbreviations DNase t-deoxyribonuclease I; F-actin-filamentous actin; PIPZ-phosphatidylinositol 4,5-bisphosphate. 0 Current Biology Ltd ISSN 0955-0674 87