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  current news   Press   selected story    
     
  7th April 2010  
 

Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

 
 




Author
Maria Hernandez-Valladares1,3, Taekyung Kim2,3, Balakrishnan Kannan1, Alvin Tung1, Adeleke H Aguda1, Mårten Larsson1, John A Cooper2 and Robert C Robinson1

1 – Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore
2 – Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri, USA.
3 – These authors contributed equally to this work.

Abstract
Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments.

 
 

 
 


Figure Legend: Structural, bioinformatics and microscopic characterization of CARMIL CPI motif interaction with capping protein (CP).
a) Structure of CARMIL CPI motif in complex with CP. CARMIL CPI motif is shown as a surface (yellow) and CP α-subunit (red) and β-subunit (blue) as schematic representations. A cartoon representation, depicting the mushroom-like CP structure with the CPI motif half-encircling the stalk on the underside of the mushroom cap, is shown on the right. (b-c) Domain organization of proteins containing CPI-motif. (b) Known CPI family members. L, leucine-rich repeat; SH3, src homology domain 3; PH, pleckstrin homology domain, CC, coiled coil. The green bars above CD2AP, CIN85 and CKIP-1 signify reported CPI regions. Black bars above CARMIL detail the constructs used in the study. (c) Novel CPI family members. WASHCAP contains multiple repeats homologous to LF(E/D)nLF (green) that shows a high incidence of serine residues. The LF(E/D)nLF motifs are generally followed by basic residue-rich regions. (d) Real-time observation of CARMIL induced uncapping of CP-capped actin filaments using TIRF microscopy. TMR-labeled (red) actin (2 μM) was polymerized and capped with CP (chicken α1/β1, 50 nM), stabilized with phalloidin and diluted to 50 nM. CBR115 was added to the CP-capped red actin filaments concurrently with fluorescein-labeled (green) actin (0.2 μM) and CP (50 nM). Profilin was maintained throughout at a 1.5 molar ratio over actin. Time-stamps (min:sec) are relative to addition of CBR115. "__" means before addition. Field-of-view is 20 x 20 μm2.

Published online in Nature Structural & Molecular Biology:
28 March 2010 | doi:10.1038/nsmb.1792

http://www.nature.com/nsmb/journal/vaop/ncurrent/pdf/nsmb.1792.pdf

For more information on Robert Robinson’s lab, please click here.