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  current news   Press   selected story    
     
  14 November 2014  
  The structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization
 
 




Authors
Bo Xuea,1,2, Cedric Leyratb,1, Jonathan M. Grimesb,c, Robert C. Robinsona,d

a  Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research),    Biopolis, Singapore 138673.
b  Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Oxford, OX3 7BN, United    Kingdom.
c   Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE,    United Kingdom.
d  Department of Biochemistry, National University of Singapore, Singapore 117597.
1  B.X. and C.L. contributed equally to this work.
2 Corresponding authors

Published in PNAS on 13 October 2014.

Abstract
Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.

Figure Legend: Tβ4 binds actin and exchanges its bound actin with profilin. (A) Domain diagrams of (Upper) the hybrid constructs of P. pastoris actin–Tβ4 and (Lower) the peptide consisting of Tβ4 and the lysine-rich region of Cordon-bleu (Cobl). (B) Structures of (Left) the Pichia actin–Tβ4 hybrid and (Right) the Tβ4–Cobl peptide in complex with rabbit skeletal muscle actin. (C) Competition between profilin and Tβ4 visualized by TIRF microscopy. (D) Binding of BODIPY-TMR–labeled full-length or N-terminally truncated Tβ4 peptides to (Left) actin or (Right) profilin:actin quantified by fluorescence anisotropy.

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