Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

Ball, Linda; Kühne, Ronald; Hoffmann, B.; Häfner, Angelika; Schmieder, Peter; Volkmer, Rudolf; Hof, Martin; Wahl, Martin A.; Schneider‐Mergener, Jens; Walter, Ulrich; Oschkinat, Hartmut; Jarchau, Thomas

doi:10.1093/emboj/19.18.4903

Cited by 129 publications

(159 citation statements)

References 47 publications

(85 reference statements)

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“…We know of two such attachments (i) via VASP and (ii) via the Arp2͞3 complex. VASP binds to the sides of filaments with 10 nM affinity (36) and to ActA with Ϸ10 M affinity (37). The Arp2͞3 complex binds to the pointed ends of new filaments with 10-50 nM affinity (11) and to full-length ActA with an affinity of 0.6 M (29).…”

Section: Bound Nucleotide Affects the Affinity Of The Arp2͞3 Complex mentioning

confidence: 99%

Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments

Dayel

Holleran

Mullins

2001

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

107

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The Arp2͞3 complex, a seven-subunit protein complex containing two actin-related proteins, Arp2 and Arp3, initiates formation of actin filament networks in response to intracellular signals. The molecular mechanism of filament nucleation, however, is not well understood. Arp2 and Arp3 are predicted to bind ATP via a highly conserved nucleotide-binding domain found in all members of the actin superfamily and to form a heterodimer than mimics a conventional actin dimer. We show here that adenosine nucleotides bind with micromolar affinity to both Arp2 and Arp3 and that hydrolyzable ATP is required for actin nucleation activity. Binding of N-WASP WA increases the affinity of both Arp2 and Arp3 for ATP but does not alter the stoichiometry of nucleotides bound in the presence of saturating concentrations of ATP. The Arp2͞3 complex bound to ADP or the nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments, but addition of the phosphate analogue BeF 3 partially restores activity to the ADP-Arp2͞3 complex. Bound nucleotide also regulates the affinity of the Arp2͞3 complex for its upstream activators N-WASP and ActA. We propose that the active nucleating form of the Arp2͞3 complex is the ADP-P i intermediate in the ATPase cycle and that the ATPase activity of the Arp2͞3 complex controls both nucleation of new filaments and release of the Arp2͞3 complex from membraneassociated activators.T he actin cytoskeleton determines the shape, motility, and internal organization of eukaryotic cells. Many actin-based structures, especially those involved in membrane protrusion, are assembled by coordinated polymerization and crosslinking of new actin filaments from actin monomers into either orthogonal or parallel filament networks (1). In these structures, work is accomplished by the free energy of polymerization. Subsequent ATP hydrolysis by filamentous actin then allows the networks to be disassembled. The rapid and regulated assembly and disassembly of actin filament networks lies at the heart of many cellular processes that involve membrane protrusion such as cell locomotion, endocytosis, and phagocytosis (2).The Arp2͞3 complex plays a central role in the regulated assembly of actin-based structures. The Arp2͞3 complex nucleates formation of new actin filaments in response to upstream signaling events and simultaneously crosslinks them into orthogonal networks. Activation of Rho-family G proteins, including Rac and Cdc42, leads to dramatic reorganization of the actin cytoskeleton (3, 4). Rac and Cdc42 promote activation of members of the WASP family of proteins (5-7) that include WASP, N-WASP, and several isoforms of Scar (8). WASP family proteins, in turn, recruit and activate the Arp2͞3 complex (7, 9, 10). The Arp2͞3 complex nucleates formation of new actin filaments from the sides of older filaments, creating a dendritic network of crosslinked actin filaments in vitro (11,12) and in vivo (13).We proposed that activation of Arp2͞3 complex involves the two actin-related proteins, Arp2 and Arp3, forming a heterodim...

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Section: Bound Nucleotide Affects the Affinity Of The Arp2͞3 Complex mentioning

confidence: 99%

Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments

Dayel

Holleran

Mullins

2001

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

107

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“…The mutant protein ActA(F351D), in which the phenylalanine of the fourth FP 4 motif was replaced by aspartic acid, can only bind three Mena EVH1 domains, indicating that phenylalanine is also critical for EVH1 binding in natively folded ActA. Amino acid residues flanking the FP 4 motif that participate in the ActA-EVH1 interaction (44,45) were not able to compensate for the mutation of phenylalanine in the fourth FP 4 motif. Mitochondria targeting assays using the human focal adhesion protein zyxin (46), which also displays four potential Ena/VASP binding sites, similarly indicate that recruitment of Mena by zyxin can only be prevented by inactivating all four FP 4 motifs through Phe3 Ala substitution.…”

Section: Acta Contains Four Equivalent Moderately Strong Binding Sitmentioning

confidence: 99%

“…Even in an extended ␤-strand conformation, IRR 2 (6 amino acid residues) and IRR 3 (5 amino acid residues) of zyxin would hardly reach a length of 13 Å necessary for simultaneous EVH1 binding. The last two FP 4 motifs of zyxin are proposed to share some flanking residues leading to a partial overlap of the EVH1-binding sites (45). Simultaneous binding of four Ena/VASP monomers or tetramers to zyxin therefore seems unlikely due to sterical hindrance of these molecules, although peptide library scans indicate that all four FP 4 motifs of zyxin are independently recognized by VASP (46).…”

Section: Acta Contains Four Equivalent Moderately Strong Binding Sitmentioning

confidence: 99%

“…Simultaneous binding of four Ena/VASP monomers or tetramers to zyxin therefore seems unlikely due to sterical hindrance of these molecules, although peptide library scans indicate that all four FP 4 motifs of zyxin are independently recognized by VASP (46). In addition, Ena/VASP proteins bind FP 4 motifs of zyxin and vinculin significantly weaker than the corresponding motifs in ActA, possibly to allow for a more regulated actin filament formation (21,26,45). Intracellular Listeria cells can thus compete successfully with the host cell proteins zyxin and vinculin for EVH1 binding for two reasons as follows: (i) the FP 4 motifs in ActA adopt a consensus sequence ideally optimized for EVH1 interaction and are, therefore, able to remove efficiently Ena/VASP proteins from their cellular localization, and (ii) the probability of Ena/VASP binding to ActA is strongly (26).…”

Section: Acta Contains Four Equivalent Moderately Strong Binding Sitmentioning

confidence: 99%

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ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Machner¹,

Urbanke²,

Barzik³

et al. 2001

Journal of Biological Chemistry

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The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP 4 ). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP 4 sites have roughly equivalent affinities with dissociation constants of about 4 M. Mutational analysis of the FP 4 motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.

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“…In vertebrates, the family is composed of VASP, mammalian Enabled (Mena), and Ena-VASP-like (EVL) (10)(11)(12), whereas invertebrates such as Caenorhabditis elegans, Drosophila melanogaster, or Dictyostelium discoideum express only one variant. All of these proteins share a conserved architecture consisting of an N-terminal Ena/VASP homology 1 (EVH1) domain that binds FP 4 motif-containing proteins for subcellular targeting (13). The central proline-rich region binds to the actin-binding protein profilin (PFN) and Src homology 3 (SH3) domains (14)(15)(16).…”

mentioning

confidence: 99%

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

Brühmann

Ushakov

Winterhoff

et al. 2017

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

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Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASPmediated actin assembly in clustered arrays.actin | Ena/VASP | TIRF | cluster | formin

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Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

Cited by 129 publications

References 47 publications

Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments

Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments

ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

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