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Ca-binding by domain 2 plays a critical role in Gelsolin activation and stabilization
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Department of Chemistry and Centre for Blood Research, Life Sciences Institute, The University of British Columbia, Canada.
Institute of Molecular and Cell Biology, A*STAR, Singapore.
Institute of Molecular and Cell Biology, A*STAR, Singapore.
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(English)Manuscript (Other (popular science, discussion, etc.))
Abstract [en]

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those previously determined for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. Thermal denaturation studies and actin depolymerization assays indicate that only two Ca-binding events are required for gelsolin activation and filament disassembly. In-depth comparison of the Ca-free and Ca-activated actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca2+ and responsible for opening the G2-G6 latch to expose the F-actin binding site on G2. Examination of Ca-binding by G2 in human G1-G3/actin reveals that the Ca2+ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 also indicate that Ca-binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidsis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca-binding by G2 prolongs the lifetime of partially activated intermediate conformations in which the protease cleavage site is exposed.

URN: urn:nbn:se:uu:diva-98740OAI: oai:DiVA.org:uu-98740DiVA: diva2:201091
Available from: 2009-03-03 Created: 2009-03-03 Last updated: 2010-01-14
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 427
National Category
Structural Biology
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)
Available from: 2009-03-02 Created: 2009-02-18 Last updated: 2010-01-13Bibliographically approved

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