Actin Hydrophobic Loop 262–274 and Filament Nucleation and Elongation

Shvetsov, Alexander; Galkin, Vitold E.; Orlova, Albina; Phillips, Martin; Bergeron, Sarah E.; Rubenstein, Peter A.; Reisler, Emil

doi:10.1016/j.jmb.2007.10.076

Cited by 17 publications

(20 citation statements)

References 32 publications

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“…This result correlates with the observation that yeast actin fragments to a greater extent than does muscle actin (4), perhaps resulting from less stable or more flexible monomer-monomer interfaces that are manifested by increased exposure to solvent. A similar association between increased filament flexibility and increased tendency to filament fragmentation was also made in a study of the binding of cofilin to F-actin (30). The results are also consistent with the work done previously with electron microscopy and three-dimensional reconstruction showing that the yeast actin filament has a nucleotide binding cleft that appears more open than that in the muscle filament and that the yeast filament has less extensive contacts between the two long pitch strands (31).…”

Section: Discussionsupporting

confidence: 89%

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

Journal of Biological Chemistry

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Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G-and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1-3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with ␤-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could ex...

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Section: Discussionsupporting

confidence: 89%

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

Journal of Biological Chemistry

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“…Because Gly residues are generally important to the conformational dynamics of proteins, and because Gly-268 is completely conserved among actin sequences, Gly-268 may contribute to the conformational dynamics of the hydrophobic loop. Conformational changes of the hydrophobic loop are unnecessary for ATPase activity accelerated under the polymerization condition (46). This is consistent with the high ATPase activity of G268D actin.…”

Section: R95c E226k G268dsupporting

confidence: 82%

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Noguchi

Gomibuchi

Murakami

et al. 2010

Journal of Biological Chemistry

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Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin⅐gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca 2؉ . Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca 2؉ sensitivity in the presence of tropomyosin-troponin.Actin filaments are major components of the cytoskeletons of all eukaryotic cells and play key roles in a variety of cellular functions, including cell migration, organelle transport, and muscle contraction. These diverse processes depend on the dynamic nature of actin filaments and their interactions with various actin-binding proteins (1), which are thought to involve conformational changes in the subunits that make up actin filaments. For instance, the stability of actin filaments is affected by the conformational difference between actin subunits carrying ATP or ADP and those that do not (2, 3), whereas cofilin, a major actin filament severing protein, alters the twist of actin filaments (4). In addition, modification of actin filaments through chemical cross-linking causes motility defects with myosin, suggesting a possible link between actin-myosin interaction and the conformational dynamics of actin (5, 6). Moreover, the myosin-induced conformational changes appear to be cooperative (7-9), e.g. Ca 2ϩ -triggered conformational changes in the skeletal muscle actin filaments are enhanced by cooperative conformational changes induced by myosin (10). In many cases, however, details of the molecular relationship between conformational changes in actin and its physiological function remain unclear.Actin is a highly conserved protein, so that Dictyostelium actin 15 shares 91% amino acid identity with rabbit skeletal muscle actin. This implies ...

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“…We fi tted the high-resolution Twf-C -G-actin to be important for filament growth and stability ( Shvetsov et al, 2008 ). In our model, the structural rearrangement of the actin subdomain 2 in the fi lament decorated with the ADF-H domain ( Fig.…”

Section: Interaction Of Twf-c and Adf/cofi Lins With Actin Fi Lamentsmentioning

confidence: 99%

Structure of the actin-depolymerizing factor homology domain in complex with actin

Paavilainen

Oksanen

Goldman

et al. 2008

The Journal of Cell Biology

150

176

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Abbreviations used in this paper: ADF-H, actin-depolymerizing factor homology; NBD, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole; WH2, WASP homology 2.The online version of this paper contains supplemental material. IntroductionPolymerization of actin fi laments against membranes produces pushing forces that are required for various cellular processes such as motility, morphogenesis, and endocytosis ( Pollard and Borisy, 2003 ;Kaksonen et al., 2006 ). Despite the large number of proteins regulating actin dynamics, many of them interact with actin through a relatively small number of protein domains. Among the central actin-binding domains is the actin-depolymerizing factor homology (ADF-H) domain, which occurs in five functionally distinct classes of proteins: ADF/cofilin, twinfi lin, Abp1/drebrin, coactosin, and glia maturation factor ( Paavilainen et al., 2007 ).The founding member of this family, ADF/cofi lin, binds both monomeric and fi lamentous actin, preferably in the ADPbound form, and induces a structural rearrangement in the actin fi lament that leads to its disassembly. When bound to an actin monomer, ADF/cofi lin inhibits spontaneous nucleotide exchange ( Carlier et al., 1997 ;Bamburg, 1999 ;Andrianantoandro and Pollard, 2006 ). In cells, ADF/cofi lin plays an essential role in various processes by promoting disassembly of aged actin fi laments ( Okreglak and Drubin, 2007 ). In contrast to ADF/cofi lin, which consists of a single ADF-H domain, twinfi lin is composed of two ADF-H domains separated by a short linker region . Twinfi lin binds ADP-actin monomers and fi lament barbed ends with high affi nity, and prevents monomer assembly into fi lament ends Helfer et al., 2006 ). In addition, yeast twinfi lin induces fi lament severing at a low pH ( Moseley et al., 2006 ). Biochemical studies suggested that during barbedend capping, twinfi lin ' s N-terminal ADF-H domain interacts with the terminal actin subunit, whereas the C-terminal ADF-H domain binds to the side of an actin fi lament through a similar mechanism to that of ADF/cofi lin ( Paavilainen et al., 2007 ). The exact functions of the Abp1/drebrin, coactosin, and glia maturation factor are less well understood, although also these proteins are linked to regulation of actin dynamics ( de Hostos et al., 1993 ;Quintero-Monzon et al., 2005 ;Ikeda et al., 2006 ).Although the biochemical activities and cellular functions of ADF-H domain proteins are rapidly being uncovered, the structure of an ADF-H domain in complex with actin has not been reported. Indirect structural methods have provided controversial results, and even the binding site of this domain on actin is not known ( Wriggers et al., 1998 ;Kamal et al., 2007 ). Consequently, the structural mechanisms by which twinfi lin and ADF/cofi lin inhibit nucleotide exchange on actin monomers and how ADF/cofi lin induces fi lament depolymerization/ severing are unknown.A ctin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal re...

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Actin Hydrophobic Loop 262–274 and Filament Nucleation and Elongation

Cited by 17 publications

References 32 publications

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Structure of the actin-depolymerizing factor homology domain in complex with actin

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