Structural and Mechanistic Studies of Two Regulatory Factors in Actin Cytoskeletal Signaling: Vav and VopL

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Title: Structural and Mechanistic Studies of Two Regulatory Factors in Actin Cytoskeletal Signaling: Vav and VopL
Author: Yu, Bingke
Abstract: Proper control of actin cytoskeletal dynamics is essential for cell survival . The goals of my thesis work have been to characterize the structural and biophysical properties of two regulatory proteins in actin cytoskeletal rearrangement pathway : Vav and Vibrio outer protein L (VopL ) . Vav proteins are guanine nucleotide exchange factors for Rho family GTPases . They play key roles in actin regulatory pathways and control diverse cellular processes like T cell maturation and activation , cell migration and phagocytosis . They belong to a group of multi -domain signaling proteins which display complex behaviors because of the collective regulation from multiple domains . Previous work has shown that Vav is autoinhibited in the resting state through the cooperative suppression of N -terminal Calponin domain and Acidic region , with the physical mechanism yet to be determined . Here through structural , energetic and biochemical studies , I demonstrate that the Calponin homology domain of Vav binds to the Pleckstrin homology domain , restrains the inhibitory helix in the Acidic region , and shifts the Dbl homology domain - inhibitory helix equilibrium to a more closed state . This construction enables strong suppression and an efficient activation process . The energetic basis of Vav autoinhibition may turn out to be widespread in multi -domain systems . VopL , a pathogenic effector from Vibrio parahaemalyticus , is an actin nucleation factor that induces stress fibers during bacterial infection . It contains three N -terminal Wiskott -Aldrich Homology 2 (WH2 ) motifs and a unique VopL C -terminal domain (VCD ) . It potently promotes actin filament nucleation in vitro . However , the physical basis of VopL mediated nucleation has not been understood . Here I performed structural and biochemical studies to investigate the mechanism of actin filament nucleation by VopL . I found that both the WH2 element and VCD are required for VopL activity . The crystal structure of VCD revealed a U -shaped dimer that is stabilized by a terminal coiled -coil . Dimerization of the WH2 motifs as well as contacts between VCD and actin contribute to the nucleation activity of VopL . My studies suggest the formation of a structurally organized actin cluster involving lateral contacts during nucleation . Stabilization of these lateral contacts may be a common feature of actin filament nucleation by WH2 -based factors .
URI: http : / /hdl .handle .net /2152 .5 /960
Date: 2011-12-15

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Structural and Mechanistic Studies of Two Regulatory Factors in Actin Cytoskeletal Signaling: Vav and VopL. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /960 .

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