ACTIN FILAMENTS DECORATED WITH CYTOSKELETAL PROTEINS

D. Hanein, S. Goldsmith*, W. Lehman**, R. Craig ***, I. Correia****, P. Matsudaira****, D.J. DeRosier, and S.C. Alma*

The W.M. Keck Inst. for Cellular Visualization, Brandeis Univ., Waltham MA 02254 *Dept. Biochem., Albert Einstein Coil. Med., Bronx, NY 10461 **Dept. Physiol., Boston University School of Medicine, Boston MA 02118 ***Dept. Cell Biol.,Univ. Mass. Med. School, Worcester MA 01655 ****Whitehead Institute far Biomedical Research, Cambridge, MA 02142 A superfamily of actin crosslinking proteins, the calponin-homology domain (CH) family is implicated in directing the assembly and controlling the organization of the cytoskeleton through a highly conserved actin binding domain (ABD). This domain, composed of a tandem pair of CH motifs, is found in actin crosslinking proteins such as fimbrin, alpha-actinin, spectrin, and dystrophin. In addition to its role in crosslinking actin filaments, a putative homologous ABD in intermediate filament-binding proteins, plectin, dystonin, and BPAGlnl, suggests a possible role in crosslinks between desmosomal and vimentin intermediate filaments and actin. Thus, the CH domain could integrate the functions of the actin and IF cytoskeletons by physically linking these structural systems. We present an atomic model for the complex of the CH domain and actin.

In crosslinking proteins that organize actin into two and three dimensional networks or gels , the ABDs lie at the ends of long, flexible molecules. Fimbrin is unique in this family, as the two ABD are tandemly repeated on the same polypepytide. The close proximity of the two ABD in fimbrin results in the formation of tight bundles instead of gels. Actin bundles found in the microvilli of the intestinal brush border epithelium and in the stereocilia of the inner ear contain the bundling protein, fimbrin. To understand such actin bundles, we need to determine the bonding rules that specify the organization of the actin filaments. Polymorphism in filament organization i.e. the absence of crystallinity, in bundles in vivo makes structural studies difficult. We are taking a two-part approach to solving the bundle structure. In the first part we generate an atomic model of actin-fimbrin (ABD) complex by: a. solving the atomic structure of fimbrin-ABD using x-ray analysis [ 11. b, producing a 28A 3D map of actin filaments decorated with the ABD using electron cryo-microscopy and image reconstruction f2]. c. docking the atomic models of fimbrin and actin into the 3D maps of the complex [33. In the second part, we obtain images of 2D arrays of actin filaments crosslinked by intact fimbrin and use the atomic model to interpret the features in the array. By electron cryo-microscopy, we determined the 3D maps of actin filaments decorated with actin-binding domain from fimbrin (N375) and of undecorated actin filaments [2]. To identify the ABD, we computed a difference map between them. The N375 difference peak is located at the same place (within our resolution limits) as the difference peak of the homologous ABDs namely the ABD of a-actinin [4] and of calponin [5]. A second difference peak has been observed upon the subtraction of F-actin from actin decorated with fimbrin. This peak was interpreted as a conformational change in actin 121. The same peak has been also found independently in studies of actin decorated with calponin [S1. To provide an atomic model of the CH-a&in complex, atomic models for the N-terminal pair of CH-domains in timbrin (101-375) [1] and for actin [6] were docked into a 28A three- dimensional map of fimbrin (N375)-actin complex. N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. The conformational change of actin seen in the difference maps appears to be centered on the C- terminal a-helix in subdomain 1. Although the ABD is composed of two CH domains (which are structurally very similar), the docking shows the two CH domains do not interact equivalently with actin. Instead one is the main site and the second interacts tangentially. The correspondence between the location of the calponin derived density, the location of the N- terminal CH domain of fimbrin, and identical conformational changes in actin are strong evidence that fimbrin, calponin, and other CH domain superfamily members bind actin through identical mechanisms.

The atomic model provides half of the information about the structure of an F-actin crosslink. In a crosslink made by fimbrin, actin filaments are separated by 140 A (center-to-center spacing) [7]. Our results show that half of this space is occupied by N375 leaving sufftcient space for the C-terminal ABD domain. The fit of the crystal structure to the difference peak suggests a model for the spatial arrangement of fimbrin domains in which tbe N-terminal ABD lies between an actin filament and the C-terminal ABD of fimbrin. The major contacts of the N-terminal ABD to the actin filament are through the N-terminal CH domain. In the x-my structure of the intact ABD, the N- and C-termini are directed in opposite orientations. Thus the C-terminal ABD of timbrin must be on the other side of N37.5 from actin and therefore in position to bind a different actin tilament

We now turn to the question of the position of the C-terminal ABD and its binding site on actin. To address this, we obtained images of 2D arrays of ~-a&in filaments crosslinked with whole Human T-fimbrin. The restriction of the filament army to two dimensions simplifies image analysis, permitting visualization of individual cross bridges. We are currently in the process of analyzing the images, i.e. determination of the spatial relationships between the filaments and their crosslinking proteins. Understanding the rules of 2D assembly and crosslinking may elucidate the 3D structure of actin-timbrin bundles.

Fig. 1: Docking of the crystal structure (black) to the timbrin (N375) difference map (white grid) [3]. The atomic model of F-actin [6] is in grey. The actin filament is the pointed end up. The F-actin diameter is approximately lOOA. Fig.2: Actin arrays crosslink with Human T-timbrin.

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