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The crystal structure of the C-terminus of adseverin: Implications for actin binding
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Robert Robinson)
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Institute of Molecular and Cell Biology, A*STAR, Singapore.
epartment of Chemistry and Centre for Blood Research, Life Sciences Institute, The University of British Columbia, Canada.
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(English)Manuscript (Other (popular science, discussion, etc.))
Abstract [en]

Adseverin is a member of the calcium-regulated gelsolin superfamily of actin severing and capping proteins. Adseverin comprises six homologous domains (A1-A6) which share 60% homology with the six domains from gelsolin (G1-G6). Adseverin is truncated in comparison to gelsolin, lacking the C-terminal extension which masks the F-actin binding site in calcium-free gelsolin. Biochemical assays have indicated differences in the interaction of the C-terminus halves of adseverin and gelsolin with actin. Gelsolin contacts actin through a major site on G4 and a minor site on G6, while adseverin uses a site on A5. Here we present the X-ray structure of the activated C-terminal half of adseverin (A4-A6). This structure is highly similar to that of the activated form of the C-terminal half of gelsolin (G4-G6), both in arrangement of domains and in the three bound calcium ions. Comparative analysis of the actin-binding surfaces observed in the G4-G6/actin structure suggests that adseverin in this conformation will also be able to interact with actin through A4 and A6, while the A5 surface is obscured. A model of calcium-free adseverin constructed from the structure of gelsolin predicts that the interaction between A2 and A6 provides sterric inhibition to prevent interaction with F-actin in the absence of calcium. Actin-binding assays reveal that the minimal stoichiometry of adseverin to calcium needed to disassemble actin filaments is 1:1 as compared to the 1:2 that was previously observed for gelsolin. We propose that the absence of a gelsolin-like C-terminal extension in adseverin reduces the calcium requirement for activation.

Keyword [en]
gelsolin superfamily proteins, gelsolin, adseverin scinderin, actin, calcium, calcium-activation
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-89393OAI: oai:DiVA.org:uu-89393DiVA: diva2:160259
Available from: 2009-02-12 Created: 2009-02-12 Last updated: 2010-01-14Bibliographically approved
In thesis
1. The Structural Basis of the Control of Actin Dynamics by the Gelsolin Superfamily Proteins
Open this publication in new window or tab >>The Structural Basis of the Control of Actin Dynamics by the Gelsolin Superfamily Proteins
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Rearrangement of the actin cytoskeleton occurs in a variety of cellular processes and structures and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins (GSPs) control actin organization by severing filaments, capping filament ends and bundling filaments. Structural changes within the GSPs are key in controling their functions. This thesis is aimed in understanding the activation mechanisms of the C-terminal halves of GSPs through investigating the atomic structures of gelsolin, adseverin and villin. X-ray crystallography was used to determine the structures of C-terminal fragments of these 3 proteins. The results demonstrate that: 1) The structure of the activated form of the C-terminal half of gelsolin displays an open conformation, with the actin-binding site on gelsolin domain 4 (G4) fully exposed and all three type-II calcium binding sites (CBS) occupied. Neither actin nor the type-I calcium, which is normally sandwiched between actin and G4, is required to achieve this conformation. 2) Calcium ions at both type-I and type-II CBSs of gelsolin were exchangable within the crystals. Extraction of calcium ions from the CBSs triggered local conformation changes which we speculate are the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. 3) The long helix of G6 in the calcium-bound structure is similar to the helix of calcium-free isolated villin domain 6 (V6). 4) The conformation of the C-terminal half of adseverin in the active state is similar to that of gelsolin. These results suggest that the C-terminal halves of GSPs are activated before forming a complex with actin. The activation involves straightening the helix of domain 6 which is a key component in the global conformation changes of C-terminal halves of these proteins. The results also suggest that a calcium ion may bind to the type-I CBS on domain 4 of the active conformation of GSPs concurrently with forming the complex with actin, hence, stabilizing the GSP:actin complex.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 37 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 425
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-89218 (URN)978-91-554-7428-7 (ISBN)
Public defence
2009-03-23, C10:305, Dept. of Medical Biochemistry and Microbiology, IMBIM, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-03-02 Created: 2009-02-09 Last updated: 2009-09-04Bibliographically approved
2. Structural and Functional Studies of Gelsolin Family Proteins
Open this publication in new window or tab >>Structural and Functional Studies of Gelsolin Family Proteins
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions including: cell locomotion, cytokinesis, chemotaxis, signal transduction and apoptosis. The coordinated assembly and disassembly of actin filaments is controlled by a multitude of proteins (ABPs) in the cell. There are over 160 actin-binding proteins known, which with actin, account for approximately 25% of cellular protein. ABPs are classified to several major groups based on their sequence identity and functions.

In this work, we have elucidated the crystal structure of ATP bound gelsolin. We have shown that ATP binding involves the two halves of gelsolin through forming numerous polar and hydrophobic contacts. Amino acid residues that form the ATP-binding sites in inactive gelsolin are widely dispersed in the activated molecule, and hence, ATP binding is disrupted on gelsolin activation. This suggests that binding of ATP may modulate the sensitivity of gelsolin to calcium ions.

The structure of human gelsolin domains 1-3 bound to actin revealed a calcium ion bound to domain 2. Here, we demonstrated that only two calcium ions are needed to activate geloslin. We speculate that this domain 2 calcium ion and the one in domain 6 participate in the initial activation of gelsolin.

The crystal structure of the activated adseverin C-terminus is highly similar to that of the C-terminus of gelsolin. Comparative analysis suggests that, like the gelsolin C-terminus, adseverin will also contact actin through domain 4 and domain 6. Biochemical experiments, presented here, show that a minimum of one calcium is required for adseverin to depolymerizing actin filaments compared to two calcium for gelsolin. We speculate that this is due to the lack of the C-terminal extension in adseverin.

We undertook a comparative analysis of four members of the gelsolin family proteins, gelsolin, adseverin, villin and capG, in the aspects of their calcium binding, pH activation and ATP binding. The results show that only gelsolin and adseverin are able to depolymerize actin filaments at pH < 6 in the absence of calcium ions and only gelsolin bind to ATP.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 41 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 427
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-98226 (URN)978-91-554-7434-8 (ISBN)
Public defence
2009-03-25, C10:301, Uppsala Biomedical Center (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-03-02 Created: 2009-02-18 Last updated: 2010-01-13Bibliographically approved

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