| 1. |
- Aguda, Adeleke H, et al.
(författare)
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The state of the filament.
- 2005
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Ingår i: EMBO Rep. - 1469-221X. ; 6:3, s. 220-6
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Tidskriftsartikel (refereegranskat)
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| 2. |
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| 3. |
- Chumnarnsilpa, Sakesit, 1967-, et al.
(författare)
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Calcium ion exchange in crystalline gelsolin
- 2006
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Ingår i: Journal of Molecular Biology. - England : Academic Press. - 0022-2836 .- 1089-8638. ; 357:3, s. 773-782
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Tidskriftsartikel (refereegranskat)abstract
- Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ion-binding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb3+, results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ion-binding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange.
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| 4. |
- Chumnarnsilpa, Sakesit, 1967-, et al.
(författare)
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The crystal structure of the C-terminus of adseverin: Implications for actin binding
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Annan publikation (populärvet., debatt m.m.)abstract
- 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.
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| 5. |
- Irobi, Edward, et al.
(författare)
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Structural basis of actin sequestration by thymosin β4 : implications for WH2 proteins
- 2004
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 23:18, s. 3599-608
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Ma, Qing, et al.
(författare)
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Ca-binding by domain 2 plays a critical role in Gelsolin activation and stabilization
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Annan publikation (populärvet., debatt m.m.)abstract
- 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.
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| 7. |
- Ma, Qing, et al.
(författare)
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Calcium, pH and ATP regulation of the gelsolin superfamily of proteins
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Annan publikation (populärvet., debatt m.m.)abstract
- CapG, adseverin, gelsolin and villin are members of the gelsolin family of calcium-sensitive regulators of actin filament dynamics. While they display some common characteristics in their interactions with and activities toward actin, they each possess unique features in their architectures and in their functions. We have probed their similarities and differences by determining the effects of Ca2+, pH, and ATP on their structures. Each undergoes conformational changes associated with activation at neutral pH when only a fraction of their potential Ca2+-binding sites is occupied. Gelsolin, villin and adseverin exhibit local minima in thermal stability in environments containing approximately 25 nM free Ca2+. The thermal stability of CapG steadily increases through this range of free Ca2+ concentrations. All four proteins share an ability to sequester actin monomers in the presence of micromolar free Ca2+, enhancing the depolymerization of F-actin. Acidic conditions at non-denaturing pH values induce a reduction in thermal stability of all four proteins. However, low pH only induces the exposure of the actin-binding sites for adseverin and gelsolin, in the absence of free Ca2+. Gelsolin alone is able to bind ATP, which stabilizes it against thermal denaturation.
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| 8. |
- Nag, Shalini, et al.
(författare)
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Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.
- 2009
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 106:33, s. 13713-8
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Tidskriftsartikel (refereegranskat)abstract
- 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 determined previously 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. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca(2+) and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca(2+) locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (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.
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| 9. |
- Nag, Shalini, et al.
(författare)
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Gelsolin : the tail of a molecular gymnast
- 2013
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Ingår i: Cytoskeleton (Hoboken, N.J.). - : Wiley. - 1949-3592 .- 1949-3584. ; 70:7, s. 360-384
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Forskningsöversikt (refereegranskat)abstract
- Gelsolin superfamily members are Ca(2+) -dependent, multidomain regulators of the actin cytoskeleton. Calcium binding activates gelsolin by inducing molecular gymnastics (large-scale conformational changes) that expose actin interaction surfaces by releasing a series of latches. A specialized tail latch has distinguished gelsolin within the superfamily. Active gelsolin exhibits actin filament severing and capping, and actin monomer sequestering activities. Here, we analyze a combination of sequence, structural, biophysical and biochemical data to assess whether the molecular plasticity, regulation and actin-related properties of gelsolin are also present in other superfamily members. We conclude that all members of the superfamily will be able to transition between a compact conformation and a more open form, and that most of these open forms will interact with actin. Supervillin, which lacks the severing domain 1 and the F-actin binding-site on domain 2, is the clear exception. Eight calcium-binding sites are absolutely conserved in gelsolin, adseverin, advillin and villin, and compromised to increasing degrees in CapG, villin-like protein, supervillin and flightless I. Advillin, villin and supervillin each contain a potential tail latch, which is absent from CapG, adseverin and flightless I, and ambiguous in villin-like protein. Thus, calcium regulation will vary across the superfamily. Potential novel isoforms of the superfamily suggest complex regulation at the gene, transcript and protein levels. We review animal, clinical and cellular data that illuminate how the regulation of molecular flexibility in gelsolin-like proteins permits cells to exploit the force generated from actin polymerization to drive processes such as cell movement in health and disease.
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| 10. |
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