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Title: Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

Abstract

Gelsolin regulates the dynamic assembly and disassembly of the actin-based cytoskeleton in non-muscle cells and clears the circulation of filaments released following cell death. Gelsolin is a six-domain (G1-G6) protein activated by calcium via a multi-step process that involves unfolding from a compact form to a more open form in which the three actin-binding sites (on the G1, G2, and G4 subdomains) become exposed. To follow the global structural changes that accompany calcium activation of gelsolin, small-angle x-ray scattering (SAXS) data were collected for full-length human plasma gelsolin at nanomolar to millimolar concentrations of free Ca{sup 2+}. Analysis of these data showed that, upon increasing free Ca{sup 2+} levels, the radius of gyration (R{sub g}) increased nearly 12 {angstrom}, from 31.1 {+-} 0.3 to 43 {+-} 2 {angstrom}, and the maximum linear dimension (D{sub max}) of the gelsolin molecule increased 55 {angstrom}, from 100 to 155{angstrom}. Structural reconstruction of gelsolin from these data provided a striking visual tracking of the gradual Ca{sup 2+}-induced opening of the gelsolin molecule and highlighted the critical role played by the flexible linkers between homologous domains. The tightly packed architecture of calcium-free gelsolin, seen from both SAXS and x-ray crystallographic models, is already partially openedmore » up in as low as 0.5 nM Ca{sup 2+}. Our data confirm that, although the molecule springs open from 0 to 1 {mu} free Ca{sup 2+}, even higher calcium concentrations help to stabilize a more open structure, with increases in R{sub g} and D{sub max} of 2 and 15 {angstrom}, respectively. At these higher calcium levels, the SAXS-based models provide a molecular shape that is compatible with that of the crystal structures solved for Ca{sup 2+}/gelsolin C-terminal and N-terminal halves {+-} monomeric G-actin. Placement of these crystal structures within the boundaries of the SAXS-based model suggests a movement of the G1/G2 subunits that would be required upon binding to actin.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930001
Report Number(s):
BNL-80612-2008-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200822%%1154
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282; Journal Issue: 35
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ACTIN; ANIMAL CELLS; AUGMENTATION; CALCIUM; CRYSTAL STRUCTURE; DEATH; FILAMENTS; HUMAN POPULATIONS; LEVELS; MOLECULES; PLASMA; PROTEINS; SCATTERING; SHAPE; SMALL ANGLE SCATTERING; X-RAY DIFFRACTION; national synchrotron light source

Citation Formats

Ashish,F., Paine, M., Perryman, P., Yang, L., Yin, H., and Krueger, J.. Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin. United States: N. p., 2007. Web. doi:10.1074/jbc.M702446200.
Ashish,F., Paine, M., Perryman, P., Yang, L., Yin, H., & Krueger, J.. Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin. United States. doi:10.1074/jbc.M702446200.
Ashish,F., Paine, M., Perryman, P., Yang, L., Yin, H., and Krueger, J.. Mon . "Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin". United States. doi:10.1074/jbc.M702446200.
@article{osti_930001,
title = {Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin},
author = {Ashish,F. and Paine, M. and Perryman, P. and Yang, L. and Yin, H. and Krueger, J.},
abstractNote = {Gelsolin regulates the dynamic assembly and disassembly of the actin-based cytoskeleton in non-muscle cells and clears the circulation of filaments released following cell death. Gelsolin is a six-domain (G1-G6) protein activated by calcium via a multi-step process that involves unfolding from a compact form to a more open form in which the three actin-binding sites (on the G1, G2, and G4 subdomains) become exposed. To follow the global structural changes that accompany calcium activation of gelsolin, small-angle x-ray scattering (SAXS) data were collected for full-length human plasma gelsolin at nanomolar to millimolar concentrations of free Ca{sup 2+}. Analysis of these data showed that, upon increasing free Ca{sup 2+} levels, the radius of gyration (R{sub g}) increased nearly 12 {angstrom}, from 31.1 {+-} 0.3 to 43 {+-} 2 {angstrom}, and the maximum linear dimension (D{sub max}) of the gelsolin molecule increased 55 {angstrom}, from 100 to 155{angstrom}. Structural reconstruction of gelsolin from these data provided a striking visual tracking of the gradual Ca{sup 2+}-induced opening of the gelsolin molecule and highlighted the critical role played by the flexible linkers between homologous domains. The tightly packed architecture of calcium-free gelsolin, seen from both SAXS and x-ray crystallographic models, is already partially opened up in as low as 0.5 nM Ca{sup 2+}. Our data confirm that, although the molecule springs open from 0 to 1 {mu} free Ca{sup 2+}, even higher calcium concentrations help to stabilize a more open structure, with increases in R{sub g} and D{sub max} of 2 and 15 {angstrom}, respectively. At these higher calcium levels, the SAXS-based models provide a molecular shape that is compatible with that of the crystal structures solved for Ca{sup 2+}/gelsolin C-terminal and N-terminal halves {+-} monomeric G-actin. Placement of these crystal structures within the boundaries of the SAXS-based model suggests a movement of the G1/G2 subunits that would be required upon binding to actin.},
doi = {10.1074/jbc.M702446200},
journal = {Journal of Biological Chemistry},
number = 35,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • No abstract prepared.
  • A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clonemore » lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.« less
  • Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1--42) and C-terminal (residues 175--270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in responsemore » to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.« less