The β-Thymosin/WH2 Domain

Hertzog, Maud; Heijenoort, Carine van; Didry, Dominique; Gaudier, Martin; Coutant, Jérôme; Gigant, B.; Didelot, Gérard; Préat, Thomas; Knossow, M.; Guittet, Éric; Carlier, Marie-France

doi:10.1016/s0092-8674(04)00403-9

Cited by 199 publications

(131 citation statements)

References 62 publications

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“…2A). The equivalent residues in ciboulot (Val-14 and Lys-31) and thymosin (Met-6 and Lys-18) sit directly in the binding interface in their respective structural models (16,31,32). In addition, our NMR data are supported by recently solved atomic structures of actin-bound WH2 domains from WASP and Scar (33).…”

Section: Discussionsupporting

confidence: 71%

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Actin Binding to the Central Domain of WASP/Scar Proteins Plays a Critical Role in the Activation of the Arp2/3 Complex

Kelly

Kranitz

Dötsch

et al. 2006

Journal of Biological Chemistry

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The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K d ‫؍‬ 10 M) than to the Arp2/3 complex (K d > 200 M). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.Rapid assembly of actin filaments is required for many basic cellular processes including amoeboid motility, endocytosis, and pathogen invasion (1, 2). One important mediator of actin assembly is the Arp2/3 complex, an essential, evolutionarily conserved, seven-subunit protein complex that nucleates and cross-links new actin filaments. The Arp2/3 complex binds the sides of pre-existing actin filaments and nucleates new filaments branching from the old at a characteristic angle of 70°(3, 4). This activity, called dendritic nucleation (4), generates space-filling networks of cross-linked and entangled actin filaments, the growth of which can generate force and power motility (5). In vitro, three factors cooperate to stimulate the nucleation activity of the Arp2/3 complex: a preexisting actin filament, an actin monomer, and a nucleation-promoting factor (NPF) 3 (6, 7). NPFs include members of the WASP/Scar family of proteins (1,8). Several WASP/Scar family proteins are regulated by small GTPases and thus serve to link Arp2/3-dependent actin assembly to upstream signals. The region of WASP/Scar family proteins that binds and activates the Arp2/3 complex is called a VCA domain and is usually located at the C terminus. A canonical VCA domain is composed of three sequences motifs: one or two V (verprolin homology) sequences (also known as WASP homology 2 or WH2 domains) that bind actin monomers; a C (central or connecting) sequence that is essential for nucleation and has been shown to interact with the Arp2/3 complex; and an A (acidi...

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Section: Discussionsupporting

confidence: 71%

“…By NMR spectroscopy we found that the WH2-actin complex is similar to the previously described ciboulot D1-actin complex (16). This result supports the proposal that the two domains are evolutionarily related (17).…”

supporting

confidence: 89%

Actin Binding to the Central Domain of WASP/Scar Proteins Plays a Critical Role in the Activation of the Arp2/3 Complex

Kelly

Kranitz

Dötsch

et al. 2006

Journal of Biological Chemistry

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“…The increased rate of exchange of the yeast versus muscle peptide again is consistent with greater solvent exposure and perhaps greater propensity of the yeast monomer to open and twist. This area is also involved in the interaction of actin with binding proteins, such as cofilin (32), and WH2 domain proteins, such as formin (33), which are involved in controlling actin filament polymerization and turnover. Different flexibility and accessibility of this region to the solvent could be important for actin isoform specificity in the interaction of actin with its binding partners.…”

Section: Discussionmentioning

confidence: 99%

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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“…The actin-bound structures of the N-terminal half of ciboulot domain 1 (9) and that of a hybrid protein consisting of gelsolin domain 1 and the C-terminal half of T␤4 (10) have been reported. These structures have been combined into a model of T␤4-actin (10), and, although both T␤4 and ciboulot belong in the T␤ family, their structures have been described as representatives of WH2 (9,10).…”

mentioning

confidence: 99%

Actin-bound structures of Wiskott–Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly

Chéreau

Kerff

Graceffa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

238

375

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Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2͞3 complex. Here we describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin ␤ domain (T␤). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than T␤ but binds actin with Ϸ10-fold higher affinity. WH2 lacks a C-terminal extension that in T␤4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2͞3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation.x-ray crystallography ͉ isothermal titration calorimetry ͉ nucleotide exchange T he actin cytoskeleton plays an essential role in many cellular functions, including intracellular transport and the control of cell shape and polarity (1). In the cell, a vast number of actin-binding proteins (ABPs) direct the location, rate, and timing for actin assembly into different structures, such as filopodia, lamellipodia, stress fibers, and focal adhesions. ABPs are commonly multidomain proteins, containing signaling domains and structurally conserved actin-binding motifs. One of the most abundant actin-binding motifs is Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) (2). The hematopoietic-specific protein, WASP, and its ubiquitously expressed ortholog N-WASP form part of a family that also includes the three WASP-family verprolin homologous protein (WAVE͞SCAR) isoforms: WAVE1, WAVE2, and WAVE3 (1, 3). Members of this family activate Arp2͞3-dependent actin nucleation and branching in response to signals mediated by Rho-family GTPases. Although the domain structure of these proteins varies, reflecting different modes of regulation, they all share a common C-terminal WH2 central-acidic region (CA region) (Fig. 1A), which constitutes the smallest fragment necessary for Arp2͞3 activation (4). WH2 is also present in members of the WASP-interacting protein (WIP) family, which form complexes with WASP͞N-WASP and modulate their functions in vivo (5, 6). Members of this family include ...

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The β-Thymosin/WH2 Domain

Cited by 199 publications

References 62 publications

Actin Binding to the Central Domain of WASP/Scar Proteins Plays a Critical Role in the Activation of the Arp2/3 Complex

Actin Binding to the Central Domain of WASP/Scar Proteins Plays a Critical Role in the Activation of the Arp2/3 Complex

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

Actin-bound structures of Wiskott–Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly

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