Proteins in the ADF/cofilin (AC) family are essential for rapid rearrangements of cellular actin structures. They have been shown to be active in both the severing and depolymerization of actin filaments in vitro, but the detailed mechanism of action is not known. Under in vitro conditions, subunits in the actin filament can treadmill; with the hydrolysis of ATP driving the addition of subunits at one end of the filament and loss of subunits from the opposite end.
View Article and Find Full Text PDFThe bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament.
View Article and Find Full Text PDFLike many bacterial pathogens, Salmonella spp. use a type III secretion system to inject virulence proteins into host cells. The Salmonella invasion protein A (SipA) binds host actin, enhances its polymerization near adherent extracellular bacteria, and contributes to cytoskeletal rearrangements that internalize the pathogen.
View Article and Find Full Text PDFTandem calponin-homology (CH) domains play an important role in the actin-binding function of many spectrin superfamily proteins. Crystal structures from several of these proteins have suggested a flexibility between these domains, and the manner in which these domains bind to F-actin has been the subject of some controversy. A recent paper has used electron microscopy and three-dimensional reconstruction to examine the complex of the utrophin tandem CH domain with F-actin.
View Article and Find Full Text PDFThe crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments.
View Article and Find Full Text PDFArthrin is a ubiquitinated actin that is present in flight muscles of some insects. In addition, it has been found in the malaria parasite Plasmodium falciparum. The role of this monoubiquitylation is not clear, and it does not appear to be associated with proteolytic degradation.
View Article and Find Full Text PDFDnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding.
View Article and Find Full Text PDFMini-chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks.
View Article and Find Full Text PDFSipA is a Salmonella protein delivered into host cells to promote efficient bacterial entry, which is essential for pathogenicity. SipA exerts its function by binding F-actin, resulting in the stabilization of F-actin and the stimulation of the bundling activity of fimbrin. Here we show that under low salt conditions where spontaneous nucleation and polymerization of actin do not occur, SipA induces extensive polymerization.
View Article and Find Full Text PDFUtrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity.
View Article and Find Full Text PDFActin is one of the most highly conserved eukaryotic proteins. There are no amino acid changes between the chicken and human skeletal muscle isoforms, and the most dissimilar actins still share more than 85% sequence identity [1]. We suggest that large discrete internal modes of freedom within the actin filament may account for a significant component of this conservation, since each subunit must make multiple specific interactions with neighboring subunits.
View Article and Find Full Text PDFSimian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag.
View Article and Find Full Text PDFNebulin is a giant protein that spans most of the muscle thin filament. Mutations in nebulin result in myopathies and dystrophies. Nebulin contains approximately 200 copies of approximately 35 residue modules, each believed to contain an actin binding site, organized into seven-module superrepeats.
View Article and Find Full Text PDF