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Title: A CapG Gain-of-Function Mutant Reveals Critical Structure and Functional Determinants for Actin Filament Severing

Abstract

CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing.

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930249
Report Number(s):
BNL-80940-2008-JA
Journal ID: ISSN 0261-4189; EMJODG; TRN: US200822%%1421
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: EMBO Journal; Journal Volume: 25
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ACTIN; AFFINITY; AMINO ACIDS; ARMS; CALCIUM; CAPACITY; CRYSTAL STRUCTURE; FILAMENTS; FORECASTING; FUNCTIONALS; FUNCTIONS; GAIN; HISTIDINE; LENGTH; MONOMERS; MUTANTS; SIMULATION; national synchrotron light source

Citation Formats

Zhang,Y., Vorobiev, S., Gibson, B., Hao, B., Dishu, G., Mishra, V., Yarmola, E., Bubb, M., Almo, S., and Southwick, F.. A CapG Gain-of-Function Mutant Reveals Critical Structure and Functional Determinants for Actin Filament Severing. United States: N. p., 2006. Web. doi:10.1038/sj.emboj.7601323.
Zhang,Y., Vorobiev, S., Gibson, B., Hao, B., Dishu, G., Mishra, V., Yarmola, E., Bubb, M., Almo, S., & Southwick, F.. A CapG Gain-of-Function Mutant Reveals Critical Structure and Functional Determinants for Actin Filament Severing. United States. doi:10.1038/sj.emboj.7601323.
Zhang,Y., Vorobiev, S., Gibson, B., Hao, B., Dishu, G., Mishra, V., Yarmola, E., Bubb, M., Almo, S., and Southwick, F.. Sun . "A CapG Gain-of-Function Mutant Reveals Critical Structure and Functional Determinants for Actin Filament Severing". United States. doi:10.1038/sj.emboj.7601323.
@article{osti_930249,
title = {A CapG Gain-of-Function Mutant Reveals Critical Structure and Functional Determinants for Actin Filament Severing},
author = {Zhang,Y. and Vorobiev, S. and Gibson, B. and Hao, B. and Dishu, G. and Mishra, V. and Yarmola, E. and Bubb, M. and Almo, S. and Southwick, F.},
abstractNote = {CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing.},
doi = {10.1038/sj.emboj.7601323},
journal = {EMBO Journal},
number = ,
volume = 25,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca/sup 2 +/ for optimal binding of actin to both sites, and for expression of its actin filament-servering function. Recent work has shown that an NH/sub 2/-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca/sup 2 +/ is present. The other binding site is Ca/sup 2 +/ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin;more » this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH/sub 2/-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca/sup 2 +/-insensitive manner. This results indicates that the NH/sub 2/-terminal half of gelsolin is the actin-severing domain. The stringent Ca/sup 2 +/ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca/sup 2 +/ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.« less
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  • In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomersmore » are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. Lastly, these phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.« less
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