A 13-A map of the actin-scruin filament from the limulus acrosomal process [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1621]

Abstract

Abstract. We have determined the structure of the actin-scruin filament to 13-/k resolution using a combination of low-dose EM and image analysis. The threedimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2÷-ADP actin filaments in the absence of Ca 2÷. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross-links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains In and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.TIS is a structural protein involved in cell motility and shape. The utility of actin lies in its abili~ to bind different actin binding proteins and generate different functional assemblies. Examples include the contractile apparatus of muscle (Huxley, 1969), microvillar bundles of intestinal epithelial cells (Maroux et al., 1988;Burgess, 1987), stereocilia of the inner ear (Flock and Cheung, 1977;DeRosier et al., 1980), the acrosomal process of Limulus sperm (Tilney, 1975), bundles within the filopodia of Dictyostelium Dharmawardhane et al., 1991;Owen et al., 1992), and the erythrocyte cytoskeleton (Branton et al., 1981). Recent findings suggest that actin structures can also provide indirect control of cellular processes. For instance, EF-la, which is an important component of protein synthesis, is also an actin bundling protein that is associated with locomotion in Dictyostelium (Yang et al., 1990;Dharmawardhane et al., 1991).Knowledge of the high resolution structure is necessary for understanding how actin can be involved in different structures and interactions. Much has been learned over the past 15 yr by EM and image analysis Vibert and Craig, 1982;Bullitt et al., 1988;Milligan et al., 1990), but the recent atomic structure of an actin:DNase I co-crystal ) is facilitating the interpretation of much of the existing data Lorenz et al., 1993;Rayment et al., 1993;Schroder et al., 1993). It allows us to consider how the subdomains of actin interact with the various actin binding proteins. Are the subdomains locked or free to move, as suggested by normal mode analysis of G-actin (Tirion and ben-Avraham, 1993)? How does the tertiary conformation change with different actin binding proteins? EM is ideally suited for looking at actin in different assemblies. Actin filaments are helical structures, and by the nature of a helix provide three-dimensional information from a single view . A resolution of 25/~ radially by 45/~ axially has been sufficient to make detailed observations of the actin filament bound with myosin subfragment-1 (S1) and tropomyosin (Milligan et al., 1987(Milligan et al...

Characterization of the Actin Cross-linking Properties of the Scruin-Calmodulin Complex from the Acrosomal Process of Limulus Sperm

“…Instead, our results suggest that scruin is bound to actin before as well as after the acrosome reaction. Although the coiled bundle of unactivated sperm has not been studied, our speculation is supported by a related structure, the supercoiled false discharge, which has been shown to contain scruin (8). Based on our studies, we propose a model that calmodulin may instead control the twist of an actin filament or bundle by regulating the conformation of scruin.…”

“…We have made major strides toward this goal through the development of methods to separate and purify native scruin from actin. Although there are several three-dimensional views of the filaments in the acrosomal process (7,8,22), this study provides the first biochemical information about the protein components of the acrosomal process. Two tricks enabled us to purify scruin as a soluble and native protein and characterize it as an actin cross-linking protein.…”

“…Thus, calmodulin bound to the neck would be flanked by a pair of actinbinding domains. We had not detected calmodulin previously because of its low molecular weight and relatively poor dyebinding capacity (6,8,23). Because the previous EM reconstructions were of acrosomes that may have been partially depleted of calmodulin (prepared in the absence of calcium), we are checking the new preparations to see if the neck region is thicker from the presence of calmodulin.…”

“…Based on sequence analysis, limited proteolysis, and EM image reconstructions, scruin is organized into two 40-kDa domains connected by a highly helical protease-sensitive neck (7)(8)(9). Each domain consists of a six-fold repeat of ϳ50 amino acids that based on the work of Bork and Doolittle (10), is predicted to fold into a four stranded ␤-sheet motif (9).…”

Effects of a Cardiomyopathy-causing Troponin T Mutation on Thin Filament Function and Structure

“…30). Resolution (31) in all reconstructions was between 2.5 and 3.0 nm; comparison of reconstructions made from images digitized on the respective scanners showed no obvious differences at this resolution. Tropomyosin and actin densities displayed in reconstructions were significant (32, 33) at equal to or greater than 99.95% confidence levels.…”