Comparison of 13Å helical and 7Å crystallographic projection maps of frozen, hydrated acrosomal bundle from limulus sperm

1995 ◽  
Vol 53 ◽  
pp. 850-851
Author(s):  
Michael F. Schmid ◽  
Joanita Jakana ◽  
Paul Matsudaira ◽  
Wah Chiu
Keyword(s):  
Extracellular Matrix ◽  
Actin Filament ◽  
Binding Sites ◽  
Repeat Unit ◽  
Nutrient Transport ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Cross Linking ◽  
3 Dimensional ◽  
Placental Protein

Actin associates with crosslinking proteins to form bundles and networks. These assemblies can provide a scaffold to anchor organelles, a support for the cell membrane or can attach the cell to the extracellular matrix or to other cells. Each crosslinker must have at least two actin binding sites, one for each actin filament. Scruin, in the Limulus acrosomal bundle, shows no homology with other actin cross-linking proteins. However, it has two homologous domains also found in the sequence encoded by kelch, a gene in Drosophila that is important in nutrient transport into the oocyte, and in MIPP, a mouse placental protein.We have determined a 13Å helical 3-dimensional structure of the actin-scruin complex, which is the basic repeat unit of the acrosomal bundle in Limulus sperm and have fitted the F-actin filament of Holmes. Each scruin binds to two adjacent actin molecules along a single F-actin filament helix. In the present investigation, we used our high resolution (7Å) spot scan electron images of ice-embedded acrosomal bundles for further analysis of the actin atomic map and scruin-scruin contacts. Spot-scan images were obtained from the frozen, hydrated acrosomal bundles in a 400 kV electron cryomicroscope.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

scholarly journals Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

1994 ◽  
Vol 126 (2) ◽  
pp. 433-443 ◽  
Author(s):  
A McGough ◽  
M Way ◽  
D DeRosier
Keyword(s):  
Image Analysis ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Outer Face ◽  
Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links

2019 ◽  
Vol 63 (3) ◽  
pp. 349-364 ◽  
Author(s):  
Sylvain D. Vallet ◽  
Sylvie Ricard-Blum
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Lysyl Oxidase ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Copper Binding ◽  
Molecular Features ◽  
Ecm Proteins ◽  
Cross Links

Abstract The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

scholarly journals Bundling of actin filaments by alpha-actinin depends on its molecular length.

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
R K Meyer ◽  
U Aebi
Keyword(s):  
Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Chicken Gizzard ◽  
Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

scholarly journals Disruption of the utrophin–actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

1998 ◽  
Vol 337 (1) ◽  
pp. 119-123 ◽  
Author(s):  
Glenn E. MORRIS ◽  
Nguyen thi MAN ◽  
Nguyen thi Ngoc HUYEN ◽  
Alexander PEREBOEV ◽  
John KENDRICK-JONES ◽  
...  
Keyword(s):  
Binding Sites ◽  
Three Dimensional ◽  
Binding Domain ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Functional Regions ◽  
Binding Surface ◽  
Actin Binding Domain ◽  
Actin Binding Site ◽  
Helical Region

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin–F-actin binding and was shown to recognize a predicted helical region at residues 13–22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.

scholarly journals The actin filament-severing domain of plasma gelsolin.

1986 ◽  
Vol 103 (4) ◽  
pp. 1473-1481 ◽  
Author(s):  
C Chaponnier ◽  
P A Janmey ◽  
H L Yin
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
The Other ◽  
Actin Binding ◽  
Plasma Gelsolin ◽  
Chymotryptic Peptide ◽  
Native Plasma

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.

scholarly journals Binding of aldolase to actin-containing filaments. Quantitative reappraisal of the interactions

1981 ◽  
Vol 195 (1) ◽  
pp. 297-299 ◽  
Author(s):  
C J Masters ◽  
D J Winzor ◽  
L W Nichol
Keyword(s):  
Theoretical Analysis ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Constant ◽  
Thin Filament ◽  
Repeat Unit ◽  
Cross Linking ◽  
A Value ◽  
Equilibrium Partition ◽  
Site Binding

Previously reported results of equilibrium-partition experiments on the interaction of aldolase with actin-containing filaments [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98] have been subjected to a more rigorous theoretical analysis involving consideration of the consequences of cross-linking interactions between enzyme and filament. The experimental results obtained with F-actin-tropomyosin are best described by a model with one binding site per heptameric repeat unit of filament and a value of 39000 M-1 for the site binding constant, k. Similar analyses of the influence of Ca2+ on aldolase binding to F-actin--tropomyosin--troponin substantiate the existence of two equivalent binding sites (k = 14900 M-1) for the enzyme on each repeat unit of the thin filament. The Ca2+-sensitivity of this interaction reflects either a decrease in the strength of aldolase binding to these two sites (k = 8200 M-1) or the elimination of one site.

Coagulation factor Va is an actin filament binding and cross-linking protein

1995 ◽  
Vol 73 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Emilia Furmaniak-Kazmierczak ◽  
Michael E. Nesheim ◽  
Graham P. Côté
Keyword(s):  
Actin Filament ◽  
Light Chain ◽  
Actin Filaments ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Actin Binding ◽  
Cross Linking ◽  
Factor V ◽  
Proteolytic Activation ◽  
Factor Va

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.

scholarly journals Twinfilin, a molecular mailman for actin monomers

2002 ◽  
Vol 115 (5) ◽  
pp. 881-886 ◽  
Author(s):  
Sandra Palmgren ◽  
Maria Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Capping Protein ◽  
Filament Assembly

Twinfilin is a ubiquitous actin-monomer-binding protein that is composed of two ADF-homology domains. It forms a 1:1 complex with ADP-actin-monomers,inhibits nucleotide exchange on actin monomers and prevents assembly of the monomer into filaments. The two ADF-H domains in twinfilin probably have 3D structures similar to those of the ADF/cofilin proteins and overlapping actin-binding sites. Twinfilin also interacts with PtdIns(4,5)P2, which inhibits its actin-monomer-sequestering activity in vitro. Mutations in the twinfilin gene result in defects in the bipolar budding pattern in S. cerevisiae and in a rough eye phenotype and aberrant bristle morphology in Drosophila melanogaster. These phenotypes are caused by the uncontrolled polymerization of actin filaments in the absence of twinfilin. Studies on budding yeast suggest that twinfilin contributes to actin filament turnover by localizing actin monomers, in their `inactive'ADP-form, to the sites of rapid filament assembly. This is mediated through direct interactions between twinfilin and capping protein. Therefore,twinfilin might serve as a link between rapid actin filament depolymerization and assembly in cells.

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