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Structural basis of actin sequestration by thymosin β4: implications for WH2 proteins
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2004 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 23, no 18, 3599-608 p.Article in journal (Refereed) Published
Description
Abstract [en]

The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

Place, publisher, year, edition, pages
2004. Vol. 23, no 18, 3599-608 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94969DOI: 10.1038/sj.emboj.7600372PubMedID: 15329672OAI: oai:DiVA.org:uu-94969DiVA: diva2:169007
Available from: 2006-10-18 Created: 2006-10-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Structural Study of the WH2 Family and Filamin: Implications for Actin Cytoskeleton Regulation
Open this publication in new window or tab >>Structural Study of the WH2 Family and Filamin: Implications for Actin Cytoskeleton Regulation
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular processes like motility, chemotaxis, phagocytosis and morphogenesis are dependent on the dynamic regulation of the actin cytoskeleton. This cytoskeleton system is tightly controlled by a number of diverse actin-binding proteins (ABPs) by various mechanisms described as nucleation, polymerization, capping, severing, depolymerization and sequestration. The ABPs are grouped based on sequence identity as in the Wiskott-Aldrich Syndrome protein homology domain 2 (WH2), and the calponin homology domain (CH) containing proteins.

In this work, we elucidate the crystal structures of hybrids of gelsolin domain 1 with thymosin β4, ciboulot domain 2, and the second WH2 domain of N-WASP each bound to actin. We show that the single WH2 motif containing protein thymosin β4 in part sequesters actin by binding its pointed end via a C-terminal helix. This interaction prevents the addition of bound actin protomers to the barbed end of the filament. We propose that sequence variations in some WH2 motifs conferred F-actin binding ability to multiple repeat-containing proteins. These F-actin binding domains interact with the barbed end of a filament and the adjacent WH2 motifs are then freed to add their bound actin to the growing filament end. We demonstrate the binding of ciboulot domains 2 and 3 to both G- and F-actin and that full length ciboulot is capable of binding two actin monomers simultaneously.

We have also cloned, expressed, purified and crystallized rod domains 14-16 from the actin crosslinking protein a-filamin. Preliminary X-ray crystallography data gives us hope that we shall be able to solve the structure of this triple domain repeat.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 182
Keyword
Biochemistry, Actin, Thymosin, Ciboulot, N-WASP, Filamin, Calponin homology domain, WH2, Protein Crystallography, Biokemi
Identifiers
urn:nbn:se:uu:diva-7188 (URN)91-554-6679-6 (ISBN)
Public defence
2006-11-08, C10:301, BMC, Hursagata 3, SE 751 23, Uppsala, 13:15
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Available from: 2006-10-18 Created: 2006-10-18Bibliographically approved

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