scholarly journals Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

1997 ◽  
Vol 8 (1) ◽  
pp. 83-95 ◽  
Author(s):  
J Prassler ◽  
S Stocker ◽  
G Marriott ◽  
M Heidecker ◽  
J Kellermann ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
Actin Isoforms ◽  
Ef Hands ◽  
Immunofluorescence Labeling ◽  
Alpha Actin

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.

scholarly journals The toxofilin–actin–PP2C complex of Toxoplasma: identification of interacting domains

2007 ◽  
Vol 401 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Gaelle Jan ◽  
Violaine Delorme ◽  
Violaine David ◽  
Celine Revenu ◽  
Angelita Rebollo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Actin Filaments ◽  
Function Analysis ◽  
Protozoan Parasite ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
The Third ◽  
Parasite Motility

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure–function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein–protein interactions that are important to parasite motility.

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.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes

1999 ◽  
Vol 112 (22) ◽  
pp. 4101-4112 ◽  
Author(s):  
K. Ojima ◽  
Z.X. Lin ◽  
Z.Q. Zhang ◽  
T. Hijikata ◽  
S. Holtzer ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thin Filament ◽  
Fluorescent Protein ◽  
De Novo ◽  
Actin Binding ◽  
Double Staining ◽  
Green Fluorescent ◽  
Short Time ◽  
Alpha Actin ◽  
De Novo Assembling

While over a dozen I-Z-I proteins are expressed in postmitotic myoblasts and myotubes it is unclear how, when, or where these first assemble into transitory I-Z-I bodies (thin filament/Z-band precursors) and, a short time later, into definitive I-Z-I bands. By double-staining the growth tips of transfected myotubes expressing (a) MYC-tagged s-alpha-actinins (MYC/s-alpha-actinins) or (b) green fluorescent protein-tagged titin cap (GFP/T-cap) with antibodies against MYC and I-Z-I band proteins, we found that the de novo assembly of I-Z-I bodies and their maturation into I-Z-I bands involved relatively concurrent, cooperative binding and reconfiguration of, at a minimum, 5 integral Z-band molecules. These included s-alpha-actinin, nebulin, titin, T-cap and alpha-actin. Resolution of the approximately 1.0 microm polarized alpha-actin/nebulin/tropomyosin/troponin thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration. Of particular interest is finding that mutant MYC/s-alpha-actinin peptides (a) lacking spectrin-like repeats 1–4, or consisting of spectrin-like repeats 1–4 only, as well as (b) mutants/fragments lacking titin or alpha-actin binding sites, were promptly and exclusively incorporated into de novo assembling I-Z-I bodies and definitive I-Z-I bands as was exogenous full length MYC/s-alpha-actinin or GFP/T-cap.

scholarly journals PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

1996 ◽  
Vol 132 (3) ◽  
pp. 359-370 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Protein Interactions ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Central Pair ◽  
Central Apparatus ◽  
Immunofluorescence Labeling ◽  
Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

NMR for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges

1998 ◽  
Vol 76 (2-3) ◽  
pp. 177-188 ◽  
Author(s):  
Jianxing Song ◽  
Feng Ni
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Experimental Approach ◽  
Structural Information ◽  
Peptide Ligands ◽  
Protein Protein Interactions ◽  
Binding Residues ◽  
Related Proteins ◽  
Binding Inhibitors

Using the design of bivalent and bridge-binding inhibitors of thrombin as an example, we review an NMR-based experimental approach for the design of functional mimetics of protein-protein interactions. The strategy includes: (i) identification of binding residues in peptide ligands by differential resonance perturbation, (ii) determination of protein-bound structures of peptide ligands by use of transferred NOEs, (iii) minimization of larger protein and peptide ligands on the basis of NMR structural information, and (iv) linkage of two weakly binding mimetics to produce an inhibitor with enhanced affinity and specificity. This approach can be especially effective for the design of potent and selective functional mimetics of protein-protein interactions because it is less likely that the surfaces of two related proteins or enzymes share two identical binding sites or regions.Key words: NMR, protein-protein interactions, functional mimetics, bridge-binding inhibitors, thrombin.

scholarly journals Elucidating the druggable interface of protein−protein interactions using fragment docking and coevolutionary analysis

2016 ◽  
Vol 113 (50) ◽  
pp. E8051-E8058 ◽  
Author(s):  
Fang Bai ◽  
Faruck Morcos ◽  
Ryan R. Cheng ◽  
Hualiang Jiang ◽  
José N. Onuchic
Keyword(s):  
Drug Design ◽  
Protein Interactions ◽  
Binding Sites ◽  
Hot Spots ◽  
Therapeutic Agents ◽  
Molecular Probes ◽  
Therapeutic Drug ◽  
Protein Protein Interactions ◽  
Protein Surface ◽  
Fragment Docking

Protein−protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein−protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

A Dynamic Escape Problem of Molecular Motors

2020 ◽  
Vol 142 (5) ◽  
Author(s):  
Dean Culver ◽  
Bryan Glaz ◽  
Samuel Stanton
Keyword(s):  
Molecular Motors ◽  
Binding Sites ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Three Dimensional ◽  
Production Capacity ◽  
Force Production ◽  
Actin Binding ◽  
Three Dimensional Model ◽  
Thermal Agitation

Abstract Animal skeletal muscle exhibits very interesting behavior at near-stall forces (when the muscle is loaded so strongly that it can barely contract). Near this physical limit, the myosin II proteins may be unable to reach advantageous actin binding sites through simple attractive forces. It has been shown that the advantageous utilization of thermal agitation is a likely source for an increased force-production capacity and reach in myosin-V (a processing motor protein), and here we explore the dynamics of a molecular motor without hand-over-hand motion including Brownian motion to show how local elastic energy well boundaries may be overcome. We revisit a spatially two-dimensional mechanical model to illustrate how thermal agitation can be harvested for useful mechanical work in molecular machinery inspired by this biomechanical phenomenon without rate functions or empirically inspired spatial potential functions. Additionally, the model accommodates variable lattice spacing, and it paves the way for a full three-dimensional model of cross-bridge interactions where myosin II may be azimuthally misaligned with actin binding sites. With potential energy sources based entirely on realizable components, this model lends itself to the design of artificial, molecular-scale motors.

scholarly journals The Disordered Cellular Multi-Tasker WIP and Its Protein–Protein Interactions: A Structural View

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1084 ◽  
Author(s):  
Chana G. Sokolik ◽  
Nasrin Qassem ◽  
Jordan H. Chill
Keyword(s):  
Protein Interactions ◽  
Cell Biology ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Actin Binding ◽  
Structural Description ◽  
Protein Protein Interactions ◽  
Intrinsically Disordered ◽  
Terminal Domain ◽  
Binding Partners

WASp-interacting protein (WIP), a regulator of actin cytoskeleton assembly and remodeling, is a cellular multi-tasker and a key member of a network of protein–protein interactions, with significant impact on health and disease. Here, we attempt to complement the well-established understanding of WIP function from cell biology studies, summarized in several reviews, with a structural description of WIP interactions, highlighting works that present a molecular view of WIP’s protein–protein interactions. This provides a deeper understanding of the mechanisms by which WIP mediates its biological functions. The fully disordered WIP also serves as an intriguing example of how intrinsically disordered proteins (IDPs) exert their function. WIP consists of consecutive small functional domains and motifs that interact with a host of cellular partners, with a striking preponderance of proline-rich motif capable of interactions with several well-recognized binding partners; indeed, over 30% of the WIP primary structure are proline residues. We focus on the binding motifs and binding interfaces of three important WIP segments, the actin-binding N-terminal domain, the central domain that binds SH3 domains of various interaction partners, and the WASp-binding C-terminal domain. Beyond the obvious importance of a more fundamental understanding of the biology of this central cellular player, this approach carries an immediate and highly beneficial effect on drug-design efforts targeting WIP and its binding partners. These factors make the value of such structural studies, challenging as they are, readily apparent.

scholarly journals Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 618-629 ◽  
Author(s):  
TJ Kovacsovics ◽  
JH Hartwig
Keyword(s):  
Actin Filaments ◽  
Myosin Ii ◽  
Surface Expression ◽  
Membrane Skeleton ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Actin Binding ◽  
Actin Assembly ◽  
Platelet Surface ◽  
Von Willebrand

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP- 280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb- IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb- IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb- IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.

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