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Structural Study of the WH2 Family and Filamin: Implications for Actin Cytoskeleton Regulation
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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. , p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 182
Keywords [en]
Biochemistry, Actin, Thymosin, Ciboulot, N-WASP, Filamin, Calponin homology domain, WH2, Protein Crystallography
Keywords [sv]
Biokemi
Identifiers
URN: urn:nbn:se:uu:diva-7188ISBN: 91-554-6679-6 (print)OAI: oai:DiVA.org:uu-7188DiVA, id: diva2:169010
Public defence
2006-11-08, C10:301, BMC, Hursagata 3, SE 751 23, Uppsala, 13:15
Opponent
Supervisors
Available from: 2006-10-18 Created: 2006-10-18Bibliographically approved
List of papers
1. Structural basis of actin sequestration by thymosin β4: implications for WH2 proteins
Open this publication in new window or tab >>Structural basis of actin sequestration by thymosin β4: implications for WH2 proteins
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2004 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 23, no 18, p. 3599-608Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-94969 (URN)10.1038/sj.emboj.7600372 (DOI)15329672 (PubMedID)
External cooperation:
Available from: 2006-10-18 Created: 2006-10-18 Last updated: 2017-12-14Bibliographically approved
2. The structural basis of actin interaction with multiple WH2/β thymosin motif-containing proteins
Open this publication in new window or tab >>The structural basis of actin interaction with multiple WH2/β thymosin motif-containing proteins
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2006 In: Structure, Vol. 14, p. 469-76Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-94970 (URN)
Available from: 2006-10-18 Created: 2006-10-18Bibliographically approved
3. Structural study of α-filamin repeats 14-16
Open this publication in new window or tab >>Structural study of α-filamin repeats 14-16
Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-94971 (URN)
Available from: 2006-10-18 Created: 2006-10-18Bibliographically approved

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