Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation

Namgoong, Suk; Boczkowska, Małgorzata; Glista, Michael J.; Winkelman, Jonathan D.; Rębowski, Grzegorz; Kovar, David R.; Domínguez, Roberto

doi:10.1038/nsmb.2109

Cited by 63 publications

(104 citation statements)

References 45 publications

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“…An interaction of the Spir proteins with the formin FH2 dimer therefore should dimerize the Spir proteins ( Figure 1). Consistent with the findings that enforced dimerization of the N-terminal Spir-WH2 domains increases Spir nucleation activity, the interaction of a Spir-KIND-WH2 protein with a nucleation incompetent C-terminal Capu dimer markedly increased the Spir nucleation activity (Quinlan et al, 2007;Namgoong et al, 2011).…”

Section: Actin Nucleation By the Spir/fmn Complexsupporting

confidence: 70%

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Structural and functional insights into the Spir/formin actin nucleator complex

Dietrich

Weiß

Pleiser

et al. 2013

Biological Chemistry

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Abstract:The diversity of cellular actin functions is attained by the activation of actin nucleator complexes, which initiate the polymerization of actin monomers into a helical double-stranded filament at defined subcellular compartments. Next to actin functions at the cell membrane, including different forms of membrane protrusions and invaginations, actin dynamics at intracellular membranes has recently become a research focus. Experiments addressing the vesicle-associated Spir WH2 domain containing actin nucleators have provided novel mechanistic insights into the function of actin dynamics at intracellular membranes. Spir proteins are targeted by a modified FYVE zinc finger motif toward endosomal and vesicle membranes, where they interact and cooperate with the distinct nucleators of the FMN subfamily of formins in the nucleation of actin filaments. The function of the Spir/ formin actin nucleator complex is closely related to the Rab11 small G protein, which is a key regulator of recycling and exocytic transport processes. Together with the actin motor protein and Rab11 effector myosin Vb, Spir/ formin nucleated actin filaments mediate actin-dependent vesicle transport processes. Drosophila and mouse genetic studies as well as cell biology experiments point toward an important role of the Spir/formin complex in oocyte maturation and in the structure and signaling of the nervous system.

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Section: Actin Nucleation By the Spir/fmn Complexsupporting

confidence: 70%

“…The Spir-WH2 nucleation rate, however, was very slow compared with the Arp2/3 complex. It was later shown that enforced dimerization of the Spir-WH2 cluster strongly enhances the nucleation activity (Namgoong et al, 2011).…”

Section: Actin Nucleation By the Spir/fmn Complexmentioning

confidence: 99%

Structural and functional insights into the Spir/formin actin nucleator complex

Dietrich

Weiß

Pleiser

et al. 2013

Biological Chemistry

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“…Despite possessing two WH2 domains, SALS-WH2 can only sequester actin monomers but cannot promote nucleation. However, the WH2 domains of SALS are similar to those found in Spire, Cordon Bleu, N-WASP or in VopL/F proteins, which sequester actin monomers, and their efficient nucleation promoting activity requires other modules surrounding the WH2 motifs (10,(12)(13)(14)(15).…”

Section: Discussionmentioning

confidence: 98%

“…The presence of tandem WH2 domains can lead to a further level of functional complexity by integrating multiple activities in actin dynamics into a single protein. In vitro, tandem WH2 domains, such as found in Spire, Cordon Bleu, N-WASP, and the Vibrio cholerae/parahemeolyticus VopL/F proteins, possess monomer sequestration activity, which can be converted into nucleation and filament depolymerizationpromoting functions by other modules surrounding the WH2 domains (10,(12)(13)(14)(15)(16)(17). Additionally, the tandem WH2 domains 2 The abbreviations used are: Tmod, tropomodulin; WH2, Wiskott-Aldrich syndrome protein homology 2; Lmod, leiomodin, SALS, sarcomere length short; IAEDANS, 1,5-IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; Alexa488NHS, Alexa Fluorா 488 carboxylic acid succinimidyl ester; TIRFM, total internal reflection fluorescence microscopy; WIP, Wiskott-Aldrich syndrome protein-interacting protein.…”

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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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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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“…Purification tags were removed (Fig. S2B), as they can potentially affect polymerization, particularly the GST tag that induces dimerization, a factor known to increase the polymerization activity of WH2 domain-based nucleators (17). Bulk polymerization was measured using the pyrene-actin assay with 2 μM Mg-ATP-actin (6% pyrene-labeled) (18).…”

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confidence: 99%

Rickettsia Sca2 has evolved formin-like activity through a different molecular mechanism

Madasu

Suarez

Kast

et al. 2013

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

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Sca2 (surface cell antigen 2) is the only bacterial protein known to promote both actin filament nucleation and profilin-dependent elongation, mimicking eukaryotic formins to assemble actin comet tails for Rickettsia motility. We show that Sca2's functional mimicry of formins is achieved through a unique mechanism. Unlike formins, Sca2 is monomeric, but has N-and C-terminal repeat domains (NRD and CRD) that interact with each other for processive barbed-end elongation. The crystal structure of NRD reveals a previously undescribed fold, consisting of helix-loop-helix repeats arranged into an overall crescent shape. CRD is predicted to share this fold and might form together with NRD, a doughnut-shaped formin-like structure. In between NRD and CRD, proline-rich sequences mediate the incorporation of profilin-actin for elongation, and WASP-homology 2 (WH2) domains recruit actin monomers for nucleation. Sca2's α-helical fold is unusual among Gram-negative autotransporters, which overwhelmingly fold as β-solenoids. Rickettsia has therefore "rediscovered" formin-like actin nucleation and elongation.passenger domain | translocator domain | spotted fever M any bacterial pathogens use the actin cytoskeleton of host eukaryotic cells for invasion and motility (1, 2). In so doing, bacteria often resort to mimicry by expressing proteins that adopt core functions of key actin cytoskeletal components, particularly actin filament nucleation and elongation factors. However, bacterial proteins tend to bypass the elaborate regulatory networks characteristic of their eukaryotic counterparts, offering a rare opportunity to dissect their functions within a simplified system (2, 3), with implications for our understanding of pathogenicity and the eukaryotic actin cytoskeleton alike.Rickettsiae are obligate intracellular Gram-negative pathogens that are transmitted to humans via arthropod vectors, such as ticks, fleas, and lice (4). Rickettsia species are responsible for a number of severe human diseases, including typhus and spotted fever (5). The spotted fever group, including Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, and over 20 other species throughout the world, uses the host-cell actin cytoskeleton to spread inter-and intracellularly. Similar to Listeria and Shigella, Rickettsia forms actin comet tails to propel its movement. However, the actin tails of Rickettsia consist of long and unbranched actin filaments, whereas those of Listeria and Shigella contain shorter and densely branched filaments (6, 7). These morphological differences stem from different molecular mechanisms for comet tail formation by these pathogens. Listeria and Shigella rely heavily on the activity of the host Arp2/3 complex that localizes uniformly along their tails (6). Although the Arp2/3 complex, activated by either host nucleation promoting factors (8) or the Rickettsia surface protein RickA (9, 10), is necessary for Rickettsia invasion (11), it is absent from Rickettsia tails (6). Another protein, Sca2 (surface cell antigen 2), has ...

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Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation

Cited by 63 publications

References 45 publications

Structural and functional insights into the Spir/formin actin nucleator complex

Structural and functional insights into the Spir/formin actin nucleator complex

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

Rickettsia Sca2 has evolved formin-like activity through a different molecular mechanism

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