Mapping the G-Actin Binding Surface of Cofilin Using Synchrotron Protein Footprinting†

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

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

Biochemical Journal ◽

10.1042/bj3370119 ◽

1998 ◽

Vol 337 (1) ◽

pp. 119-123 ◽

Cited By ~ 2

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.

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The structure of the actin filament uncapping complex mediated by twinfilin

Science Advances ◽

10.1126/sciadv.abd5271 ◽

2021 ◽

Vol 7 (5) ◽

pp. eabd5271

Author(s):

Dennis M. Mwangangi ◽

Edward Manser ◽

Robert C. Robinson

Keyword(s):

Actin Filament ◽

Protein Interactions ◽

Protein Complex ◽

Actin Filaments ◽

Actin Binding ◽

Capping Protein ◽

Binding Surface ◽

Actin Capping Protein ◽

Actin Capping

Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein–associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G-actin conformation in the two terminal actin subunits. Furthermore, twinfilin disorders critical actin-capping protein interactions, which will assist in the dissociation of capping protein, and may promote filament uncapping through a second mechanism involving V-1 competition for an actin-binding surface on capping protein. The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments.

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Single Molecule Kinetic Analysis of Actin Filament Capping

Journal of Biological Chemistry ◽

10.1074/jbc.m705287200 ◽

2007 ◽

Vol 282 (38) ◽

pp. 28014-28024 ◽

Cited By ~ 62

Author(s):

Jeffrey R. Kuhn ◽

Thomas D. Pollard

Keyword(s):

Fission Yeast ◽

Rate Constant ◽

Single Molecule ◽

Actin Filaments ◽

Total Internal Reflection ◽

Dissociation Rate ◽

Actin Binding ◽

Capping Protein ◽

Capping Proteins ◽

Binding Surface

We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 × 106m-1 s-1 and dissociation of 0.0003 s-1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), PI(4,5)P2, and PI(3,4,5)P3, prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 × 105m-1 s-1) and a faster dissociation rate constant (0.004 s-1). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.

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DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in Drosophila PRC1

10.1101/2020.06.02.130492 ◽

2020 ◽

Author(s):

Jin Joo Kang ◽

Denis Faubert ◽

Jonathan Boulais ◽

Nicole J. Francis

Keyword(s):

Mass Spectrometry ◽

Dna Binding ◽

Ubiquitin Ligase ◽

Terminal Region ◽

Cross Linking ◽

Chromatin Binding ◽

Protein Footprinting ◽

Intrinsically Disordered ◽

The Core ◽

Binding Surface

AbstractPolycomb Group (PcG) proteins regulate gene expression by modifying chromatin. A key PcG complex, Polycomb Repressive Complex 1 (PRC1), has two activities: a ubiquitin ligase activity for histone H2A, and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal homology region (HR) of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase for H2A. The intrinsically disordered C-terminal region of PSC (PSC-CTR) compacts chromatin, and inhibits chromatin remodeling and transcription in vitro. Both the PSC-HR and the PSC-CTR are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used cross-linking mass spectrometry (XL-MS) to analyze the conformations of the PSC-CTR in PRC1 and how they change on binding DNA. XL-MS identifies interactions between the PSC-CTR and the core of PRC1, including between the PSC-CTR and PSC-HR. New contacts and overall more compacted PSC-CTR conformations are induced by DNA binding. Protein footprinting of accessible lysine residues in the PSC-CTR reveals an extended, bipartite candidate DNA/chromatin binding surface. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible PSC-CTR. Intramolecular interactions of the PSC-CTR detected by XL-MS can bring the high affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large IDRs that bind nucleic acids.HighlightsAn intrinsically disordered region (IDR) in Polycomb protein PSC compacts chromatinCross-linking mass spectrometry (XL-MS) was used to analyze topology of the PSC IDRProtein footprinting suggests a bipartite DNA binding surface in the PSC IDRA model for the DNA-driven organization of the PSC IDRCombining XL-MS and protein footprinting is a strategy to understand nucleic acid binding IDRsAbstract Figure

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Identification of an Actin Binding Surface on Vinculin that Mediates Mechanical Cell and Focal Adhesion Properties

Structure ◽

10.1016/j.str.2014.03.002 ◽

2014 ◽

Vol 22 (5) ◽

pp. 697-706 ◽

Cited By ~ 34

Author(s):

Peter M. Thompson ◽

Caitlin E. Tolbert ◽

Kai Shen ◽

Pradeep Kota ◽

Sean M. Palmer ◽

...

Keyword(s):

Focal Adhesion ◽

Actin Binding ◽

Adhesion Properties ◽

Binding Surface

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Disruption of the utrophin‒actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

Biochemical Journal ◽

10.1042/0264-6021:3370119 ◽

1999 ◽

Vol 337 (1) ◽

pp. 119 ◽

Cited By ~ 7

Author(s):

Glenn E. MORRIS ◽

Nguyen thi MAN ◽

Nguyen thi Ngoc HUYEN ◽

Alexander PEREBOEV ◽

John KENDRICK-JONES ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Actin Binding ◽

Binding Surface

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Discovery of the fastest myosin, its amino acid sequence, and structural features

10.1101/2021.05.06.442907 ◽

2021 ◽

Author(s):

Takeshi Haraguchi ◽

Masanori Tamanaha ◽

Kano Suzuki ◽

Kohei Yoshimura ◽

Takuma Imi ◽

...

Keyword(s):

Actin Filaments ◽

Cytoplasmic Streaming ◽

Structural Features ◽

Chara Corallina ◽

Actin Binding ◽

X Ray Crystallography ◽

Binding Surface ◽

Fast Movement ◽

Myosin Xi ◽

Loop 2

Cytoplasmic streaming with extremely high velocity (~70 μm s−1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 μm s−1, the fastest myosin measured so far, exists in Chara cells. However, the previously cloned Chara corallina myosin XI (CcXI) moved actin filaments at a velocity of around 20 μm s−1, suggesting that an unknown myosin XI with a velocity of 70 μm −1 may be present in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 were similar to that of CcXI, the velocities of CbXI-1 and CbXI-2 were estimated to be 73 and 66 μm s−1, respectively, suggesting that CbXI-1 and CbXI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that CbXI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis.

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Distribution of c-protein isoforms in sarcomeres of developing chick skeletal muscle

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010010319x ◽

1988 ◽

Vol 46 ◽

pp. 226-227

Author(s):

D. A. Fischman ◽

J. E. Dennis ◽

T. Obinata ◽

H. Takano-Ohmuro

Keyword(s):

Skeletal Muscles ◽

Thick Filament ◽

Protein Isoforms ◽

Actin Binding ◽

Striated Muscles ◽

C Protein ◽

Sarcomeric Protein ◽

Thick Filaments ◽

Cross Bridges ◽

Bridge Bearing

C-protein is a 150 kDa protein found within the A bands of all vertebrate cross-striated muscles. By immunoelectron microscopy, it has been demonstrated that C-protein is distributed along a series of 7-9 transverse stripes in the medial, cross-bridge bearing zone of each A band. This zone is now termed the C-zone of the sarcomere. Interest in this protein has been sparked by its striking distribution in the sarcomere: the transverse repeat between C-protein stripes is 43 nm, almost exactly 3 times the 14.3 nm axial repeat of myosin cross-bridges along the thick filaments. The precise packing of C-protein in the thick filament is still unknown. It is the only sarcomeric protein which binds to both myosin and actin, and the actin-binding is Ca-sensitive. In cardiac and slow, but not fast, skeletal muscles C-protein is phosphorylated. Amino acid composition suggests a protein of little or no αhelical content. Variant forms (isoforms) of C-protein have been identified in cardiac, slow and embryonic muscles.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122964 ◽

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.

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