A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex

Panchal, Sanjay C.; Kaiser, Donald A.; Torres, Eduardo M.; Pollard, Thomas D.; Rosen, Michael K.

doi:10.1038/nsb952

Cited by 141 publications

(155 citation statements)

References 43 publications

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“…A combination of TROSY pulse sequences, 100% deuteration, buffer optimization (23), and use of a cryoprobe enabled us to acquire high quality NOESY spectra at protein concentrations of 150 -400 M. From the observed NOEs we determined direct connectivity of a number of residues. Consistent with previous reports (7,24), our data indicate that the VC domain by itself is unfolded and that about 40% of the VC peptide adopts a helical conformation upon binding actin.…”

Section: Secondary Structure and Interacting Residues Of The Vc-actinsupporting

confidence: 81%

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: Secondary Structure and Interacting Residues Of The Vc-actinsupporting

confidence: 81%

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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“…Therefore, a path along the actin nucleotide cleft is not necessary for nucleotide exchange inhibition, suggesting that this function resides within the N-terminal part of WH2 and T␤. In agreement with this notion, WIP [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] , corresponding to the N-terminal ␣-helix, inhibits nucleotide exchange as potently as the full-length WH2 motif after taking into consideration the lower actin-binding affinity of this peptide. Therefore, we conclude that nucleotide exchange inhibition is a function of the N-terminal ␣-helix of WH2 and T␤, which binds between actin subdomains 1 and 3, away from the nucleotide site.…”

Section: Control Of Actin Nucleotide Exchange By Wh2 and T␤supporting

confidence: 48%

“…This conclusion is further supported by the fact that T␤4 , starting from the LKKT sequence to the end, fails to bind actin. Similarly, the N-terminal portion of WIP (WIP [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] ) accounts for most of its binding affinity, whereas WIP 46-63 binds very weakly. Therefore, it can be concluded that hydrophobic contacts involving the N-terminal ␣-helices of WH2 and T␤ play a predominant role in the interaction with actin over electrostatic interactions within their C-terminal portions.…”

Section: Resultsmentioning

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.

237

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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“…Expression of GFP-A inhibited HIV-1 infection less efficiently than other derivatives (57.7%, p Ͻ 0.05; Figure 4C). However, GFP-A was unable to limit IMV infection (110.8%, Figure 4C) although synthetic peptides of A subdomain has been shown to retain the binding affinity to the Arp2/3 complex as high as VCA in vitro (Panchal et al, 2003). These data suggested that the V subdomain was not required for the inhibition of HIV-1 and IMV infection.…”

Section: Gfp-vca's Ability To Nucleate Actin Filament Is Not Necessarmentioning

confidence: 52%

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

Komano

Miyauchi

Matsuda

et al. 2004

MBoC

100

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Characterizing cellular factors involved in the life cycle of human immunodeficiency virus type 1 (HIV-1) is an initial step toward controlling replication of HIV-1. Actin polymerization mediated by the Arp2/3 complex has been found to play a critical role in some pathogens' intracellular motility. We have asked whether this complex also contributes to the viral life cycles including that of HIV-1. We have used both the acidic domains from actin-related protein (Arp) 2/3 complex-binding proteins such as the Wiscott-Aldrich syndrome protein (N-WASP) or cortactin, and siRNA directing toward Arp2 to inhibit viral infection. HIV-1, simian immunodeficiency virus (SIV), and intracellular mature vaccinia virus (IMV) were sensitive to inhibition of the Arp2/3 complex, whereas MLV, HSV-1, and adenovirus were not. Interestingly, pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) overcame this inhibition. Constitutive inhibition of the Arp2/3 complex in the T-cell line H9 also blocked replication of HIV-1. These data suggested the existence of an Arp2/3 complex-dependent event during the early phase of the life cycles of both primate lentiviruses and IMV. Inhibiting the HIV-1's ability to activate Arp2/3 complex could be a potential chemotherapeutic intervention for acquired immunodeficiency syndrome (AIDS)

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A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex

Cited by 141 publications

References 43 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-bound structures of Wiskott–Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

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