News archives


OCTOBER - DECEMBER 17

JULY - SEPTEMBER 17

APRIL - JUNE 17

JANUARY - MARCH 17

OCTOBER - DECEMBER 16

JULY - SEPTEMBER 16

APRIL - JUNE 16

JANUARY - MARCH 16

OCTOBER - DECEMBER 15

JULY - SEPTEMBER 15

APRIL - JUNE 15

JANUARY - MARCH 15

OCTOBER - DECEMBER 14

JULY - SEPTEMBER 14

APRIL - JUNE 14

JANUARY - MARCH 14

OCTOBER - DECEMBER 13

JULY - SEPTEMBER 13

APRIL - JUNE 13

JANUARY - MARCH 13

OCTOBER - DECEMBER 12

JULY - SEPTEMBER 12

APRIL - JUNE 12

JANUARY - MARCH 12

OCTOBER - DECEMBER 11

JULY - SEPTEMBER 11

APRIL - JUNE 11

JANUARY - MARCH 11

OCTOBER - DECEMBER 10

JULY - SEPTEMBER 10

APRIL - JUNE 10

JANUARY - MARCH 10

OCTOBER - DECEMBER 09

JULY - SEPTEMBER 09

APRIL - JUNE 09

JANUARY - MARCH 09

OCTOBER - DECEMBER 08

JULY - SEPTEMBER 08

APRIL - JUNE 08

JANUARY - MARCH 08

OCTOBER - DECEMBER 07

JULY - SEPTEMBER 07

APRIL - JUNE 07

JANUARY - MARCH 07

 
  current news   Press   selected story    
     
  14th September 2009  
 

Three insights into the activation of the gelsolin superfamily of actin regulatory proteins

 
 




1. Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.

Authors

Shalini Naga,1, Qing Maa,b,1, Hui Wangc,1, Sakesit Chumnarnsilpaa, Wei Lin Leea, Mårten Larssona, Balakrishnan Kannana, Maria Hernandez-Valladaresa, Leslie D. Burtnickc, and Robert C. Robinsona

a Institute of Molecular and Cell Biology, A*STAR, 61 Biopolis Drive, Proteos, Singapore 138673
b Institutionen for Medicinsk Biokemi och Mikrobiologi, Box 582, 751 23 Uppsala, Sweden
c Department of Chemistry and Centre for Blood Research, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada V6T 1Z1

1 These authors contributed equally

Published in the Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13713-8.

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 Ca2+ 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 Ca2+ 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.

 
 



 
 


Figure Legend:
Structural interdependence of the G2 and G6 Ca-binding sites. (A) Schematic and electrostatic surface representations of Ca-free human gelsolin, highlighting the charged residues at the G2-G6 interface. (B) Schematic representations of Ca-bound G6 and G2, taken from the structures of Ca-bound equine G4-G6/actin (Protein Data Bank ID 1H1V) and human G1-G3/actin, respectively, in orientations similar to those presented in A. G2 and G6 have been translated relative to their positions in A to avoid steric clashes. Note that the Ca ions are coordinated by residues that previously made up the network of interactions between G2 and G6 in A.


2. The crystal structure of the C-terminus of adseverin reveals the actin-binding interface.

Authors

Sakesit Chumnarnsilpaa,b, Wei Lin Leea, Shalini Naga, Balakrishnan Kannana, Mårten Larssona, Leslie D. Burtnickc, and Robert C. Robinsona

a Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673
b Institutionen for Medicinsk Biokemi och Mikrobiologi, Uppsala University, Box 582, 751 23 Uppsala, Sweden
c Department of Chemistry and Centre for Blood Research, Life Sciences Institute, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada

Published in the Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13719-24.

Abstract
Adseverin is a member of the calcium-regulated gelsolin superfamily of actin severing and capping proteins. Adseverin comprises 6 homologous domains (A1–A6), which share 60% identity with the 6 domains from gelsolin (G1–G6). Adseverin is truncated in comparison to gelsolin, lacking the C-terminal extension that masks the F-actin binding site in calcium-free gelsolin. Biochemical assays have indicated differences in the interaction of the C-terminal halves of adseverin and gelsolin with actin. Gelsolin contacts actin through a major site on G4 and a minor site on G6, whereas 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 3 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, whereas the A5 surface is obscured. A single residue mutation in A4–A6 located at the predicted A4/actin interface completely abrogates actin sequestration. A model of calcium-free adseverin, constructed from the structure of gelsolin, predicts that in the absence of a gelsolin-like C-terminal extension the interaction between A2 and A6 provides the steric inhibition to prevent interaction with F-actin. We propose that calcium binding to the N terminus of adseverin dominates the activation process to expose the F-actin binding site on A2.

 
 



 
 


Figure Legend:
Model of actin binding by A4-A6. The structure of A4-A6 placed on actin (green surface) in accordance with the G4-G6/actin structure. Regions of contact from A4 (schematic) and A6 (charge surface) with actin are shown magnified in comparison to the homologous regions from gelsolin. In particular, residue Asp-461 is conserved (Asp-487 gelsolin), which mediates calcium binding with actin. The yellow strand within A5, indicated by an arrow, depicts the actin nucleating fragment from A5.


3. Helix straightening as an activation mechanism in the gelsolin superfamily of actin regulatory proteins.

Authors

Hui Wangc, Sakesit Chumnarnsilpaa,b, Anantasak Loonchantac, Qiang Lid, Yang-Mei Kuana, Sylvie Robinee, Mårten Larssona, Ivana Mihalekd, Leslie D. Burtnickc, and Robert C. Robinsona

a Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673
b Institutionen for Medicinsk Biokemi och Mikrobiologi, Uppsala University, Box 582, 751 23 Uppsala, Sweden
c Department of Chemistry and Centre for Blood Research, Life Sciences Institute, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
d Bioinformatics Institute, A*STAR, 30 Biopolis Drive, Matrix, Singapore 138671
e UMR 144, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France

Published in the J Biol Chem. 2009 Aug 7;284(32):21265-9.

Abstract
Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca2+ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca2+, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca2+ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca2+, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites.



Figure Legend: Structural comparison of villin V6 (Ca2+-free) with gelsolin G6 (Ca2+-bound and Ca2+-free). (A) Schematic representation of isolated Ca2+-free V6. (B) Key residues that are involved in the putative V6 calcium-binding site, viewed from below with respect to A. (C) and (D) Ca2+ -bound form of gelsolin G6, displaying a straight helix, taken from the Ca2+-bound form of G4-G6 (Protein Data Bank code 1p8x). (E) and (F) Ca2+-free form of G6 revealing a kinked helix and a translocated AB loop, taken from the structure of whole plasma gelsolin (Protein Data Bank code 1d0n). The Ca2+-binding residues are dislocated in the absence of Ca2+ compared with D. A hydrogen bond between Asn647 and Arg702 links the bending of the helix to movement of the AB loop. Protein representations were generated here and in the figures that follow using PyMOL.


For more information on Robert ROBINSON’s Lab, Please click here.