How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly

Didry, Dominique; Cantrelle, François-Xavier; Husson, Clotilde; Roblin, Pierre; Moorthy, Anna M Eswara; Pérez, Javier; Clainche, Christophe Le; Hertzog, Maud; Guittet, Éric; Carlier, Marie-France; Heijenoort, Carine van; Renault, Louis

doi:10.1038/emboj.2011.461

Cited by 55 publications

(105 citation statements)

References 45 publications

(93 reference statements)

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“…The fact that the stoichiometry of SALS-WH2⅐G-actin depends on the location of the fluorophore, highlights the importance of the careful use of fluores- Comparative sequence analysis of WH2 domains from different proteins supports the conclusion that both WH2 domains of SALS bind to G-actin and predict a sequestering mechanism (Fig. 1A) (7). The N-terminal part of both WH2 domains of SALS possesses a conserved hydrophobic amino acid doublet/triplet composed of Met, Leu, Val, or Ile.…”

Section: Discussionmentioning

confidence: 88%

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Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein

Tóth

Majoros

Vig

et al. 2016

Journal of Biological Chemistry

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Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer-filament equilibrium toward the monomeric actin. In addition, SALS-WH2 can bind to but fails to depolymerize phalloidin-or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with the following two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.The basic myofibrillar contractile units, sarcomeres, orchestrate the function of striated muscle. Essential components of the sarcomeric protein networks are the thin filaments composed of actin and regulatory proteins. During sarcomerogenesis, the assembly of actin monomers into filaments and the organization of these filaments into higher order complexes result in the formation of thin filaments. Filament barbed ends, anchored to the Z disk by ␣-actinin, are capped by CapZ, whereas tropomodulin (Tmod) 2 caps pointed ends. Therefore, sarcomeric thin filaments with well defined length are arranged in an extremely regular manner. The structural features of sarcomeric actin filaments are well resolved; however, the mechanisms governing the assembly and proper organization of actin filaments are not completely understood.The Wiskott-Aldrich syndrome protein homology 2 (WH2) domains are found in increasing numbers of proteins that regulate the actin cytoskeleton during morphogenetic and motile processes (1-6). When uncomplexed, the isolated WH2 domain is a short structurally and intrinsically disordered region in solution, which shows partial folding upon binding to actin (7,8). The central element of the domain is an LKK(T/V) consensus motif (Fig. 1A). This motif is flanked by an N-terminal amphipathic ␣-helix followed by an FXXXK linker and a C-terminal region with variable length and composition. WH2 can occur as a single module or as tandem regions within the actin-associated proteins. Interesting features of the WH2 domain...

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Section: Discussionmentioning

confidence: 88%

“…When uncomplexed, the isolated WH2 domain is a short structurally and intrinsically disordered region in solution, which shows partial folding upon binding to actin (7,8). The central element of the domain is an LKK(T/V) consensus motif (Fig.…”

mentioning

confidence: 99%

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein

Tóth

Majoros

Vig

et al. 2016

Journal of Biological Chemistry

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“…As outlined above, other formins are activated by actin-binding protein domains that act either externally (like Bud6, which activates Bni1 and also binds actin in a WH2-like fashion (35,37) or internally (like the C-terminal WH2 domain of INF2 or FMNL3 (30,31)). The model we present here for the role of FSI in the processive function of Fmn2 may thus have general significance regarding the regulation of other formins.…”

Section: Discussionmentioning

confidence: 99%

“…The binding of FSI caused a 22% increase in AEDANS-actin fluorescence, consistent with establishing a more hydrophobic environment of the probe covalently attached to cysteine 374. In contrast, binding of WH2 domains or thymosin ␤4 generally quenches the fluorescence of AEDANS-actin, by making the environment of AEDANS more polar (30,31). FSI bound G-actin in competition with thymosin ␤4 but did not compete with profilin, indicating that a ternary complex of actin with profilin and FSI formed in which the affinity of FSI (and of profilin as well) for G-actin is decreased by only 2-3-fold (Fig.…”

Section: Table 2 Thermodynamic Parameters Of Formin 2 (Fmn2) and Spirmentioning

confidence: 99%

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Montaville¹,

Kühn²,

Compper

et al. 2016

Journal of Biological Chemistry

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Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin⅐actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end.Polarized assembly of actin filaments is pivotal in motile processes. It is precisely regulated by proteins that bind the barbed ends of actin filaments and either block or assist filament assembly using various mechanisms (1). Among these regulators, formins are acknowledged to nucleate and track filament barbed ends, mediating rapid processive elongation of filaments (2, 3). In contrast, WASP homology 2 (WH2) 3 repeatcontaining proteins display versatile barbed end capping or tracking and filament severing activities (4). In early oogenesis of Drosophila as well as in mammalian meiosis, a formin (Fmn2/Cappuccino) and a WH2-repeat protein (Spire) synergize in an intriguing fashion to stimulate massive assembly of a dynamic cytoplasmic actin meshwork, required for completion of crucial steps in axis patterning or asymmetric division (5-12). This complexity adds to the known need of profilin for rapid processive assembly of all formins, including Cappuccino (9). Like most formins, Fmn2 harbors conserved C-terminal formin homology 1 (FH1) and formin homology 2 (FH2) domains. The C-terminal FH1-FH2 domain of Fmn2 represents a constitutive active module in which the FH1 domain binds profilin and the FH2 domain to the barbed end of F-actin. The head-to-tail FH2 dimer of formins is thought, from structural data, to encircle the terminal and subterminal actin subunits that constitute the barbed end of the fil...

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“…Generally accepted is the subdivision of ABMs into WH2 domains, characterized by longer conserved regions preceding the amphiphilic helix, and b-thymosins, identified by longer C-terminal extended segments and a conserved linker connecting the helix and the LKKT motif [40]. Subtle sequence changes between ABMs have been shown to shift their activity from sequestering monomeric G-actin, thus inhibiting polymerization, to directing assembly of filaments (F-actin) [38,41,42]. It has also been hypothesized that ABMs mediate the interaction of actin with other actin binding proteins such as profilin [36,42].…”

Section: Introductionmentioning

confidence: 99%

New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

et al. 2015

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WASp-interacting protein (WIP) is an intrinsically disordered 503-residue polypeptide with a key role in actin polymerization in activated T cells. Its interaction with actin is mediated by a pair of conserved actin binding motifs (ABMs) at the WIP N-terminus, a domain that has not been investigated in its unbound form. Here we use NMR to investigate the biophysical behavior of the N-terminal ABM in WIP using protonless 13 C 0 -detected spectroscopy. Secondary chemical shifts, residual dipolar couplings and temperature effects identify residual structure throughout the ABM, which exhibits transient helical and b-strand character for residues 30-42 and 44-62, respectively. These observed structural propensities echo the structure observed in the actin-bound state of the ABM. Furthermore, residues preceding the canonical ABM (17-25) and conserved among WIP-related proteins exhibit transient b-strand character, suggesting that the WIP N interaction epitope extends towards the N-terminal polyproline motif. This suggests a possible role for this region in mediating the WIP interaction with polyproline binders such as profilin. In revealing these features of the WIP ABM this study demonstrates the unique ability of NMR in characterizing unstructured domains and provides necessary information for further investigation of WIP-mediated protein-protein interactions.

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How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly

Cited by 55 publications

References 45 publications

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

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