Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly
ABSTRACT The Ena/VASP and WASP family of proteins play distinct roles in actin cytoskeleton remodeling. Ena/VASP is linked to actin filament elongation, whereas WASP plays a role in filament nucleation and branching mediated by Arp2/3 complex. The molecular mechanisms controlling both processes are only emerging. Both Ena/VASP and WASP are multidomain proteins. They both present poly-Pro regions, which mediate the binding of profilin-actin, followed by G-actin-binding (GAB) domains of the WASP-homology 2 (WH2) type. However, the WH2 of Ena/VASP is somewhat different from that of WASP, and has been poorly characterized. Here we demonstrate that this WH2 binds profilin-actin with higher affinity than actin alone. The results are consistent with a model whereby allosteric modulation of affinity drives the transition of profilin-actin from the poly-Pro region to the WH2 and then to the barbed end of the filament during elongation. Therefore, the function of the WH2 in Ena/VASP appears to be to "process" profilin-actin for its incorporation at the barbed end of the growing filament. Conformational changes in the newly incorporated actin subunit, resulting either from nucleotide hydrolysis or from the G- to F-actin transition, may serve as a "sensor" for the processive stepping of Ena/VASP. Conserved domain architecture suggests that WASP may work similarly.
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ABSTRACT: Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin-actin complexes constitute the largest fraction of polymerization-competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin-actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin-actin by the last poly-Pro segment of vasodilator-stimulated phosphoprotein (VASP) and the binding of profilin-actin simultaneously to this poly-Pro and to the G-actin-binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.The EMBO Journal 11/2007; 26(21):4597-606. DOI:10.1038/sj.emboj.7601874 · 10.75 Impact Factor
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ABSTRACT: Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.Molecular Biology of the Cell 08/2007; 18(7):2579-91. DOI:10.1091/mbc.E06-11-0990 · 4.55 Impact Factor
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ABSTRACT: The adaptor protein missing-in-metastasis (MIM) contains independent F- and G-actin binding domains, consisting, respectively, of an N-terminal 250 aa IRSp53/MIM homology domain (IMD) and a C-terminal WASP-homology domain 2 (WH2). We determined the crystal structures of MIM's IMD and that of its WH2 bound to actin. The IMD forms a dimer, with each subunit folded as an antiparallel three-helix bundle. This fold is related to that of the BAR domain. Like the BAR domain, the IMD has been implicated in membrane binding. Yet, comparison of the structures reveals that the membrane binding surfaces of the two domains have opposite curvatures, which may determine the type of curvature of the interacting membrane. The WH2 of MIM is longer than the prototypical WH2, interacting with all four subdomains of actin. We characterize a similar WH2 at the C terminus of IRSp53 and propose that in these two proteins WH2 performs a scaffolding function.Structure 03/2007; 15(2):145-55. DOI:10.1016/j.str.2006.12.005 · 6.79 Impact Factor