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Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

Nakamura F, Heikkinen O, Pentikäinen OT, Osborn TM, Kasza KE, Weitz DA, Kupiainen O, Permi P, Kilpeläinen I, Ylänne J, Hartwig JH, Stossel TP - PLoS ONE (2009)

Bottom Line: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction.FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations.Disease-related FLNa mutations have demonstrable effects on FLNa function.

View Article: PubMed Central - PubMed

Affiliation: Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. fnakamura@rics.bwh.harvard.edu

ABSTRACT

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans.

Methodology/principal findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress.

Conclusions/significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

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FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP.(A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.
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pone-0004928-g002: FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP.(A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.

Mentions: FLAG-tagged full-length FLNa pulled down full-length FilGAP in vitro, and deletion of FLNa Ig repeat 23 abolished this interaction (Figure 2A), consistent with previous results [12]. The interaction was diminished or abolished respectively by deletion of hinge 2 and FLNa Ig repeat 24 (Figure 2A). Deletion of the hinge 2 from the C-terminal of IgFLNa23-24 disrupted FilGAP binding (Figure 2B). A hinge 2 plus IgFLNa24 construct does not bind FilGAP (data not shown), indicating that hinge 2 is not the FilGAP-binding site. Electron micrographs of mutant FLNa constructs demonstrate that their shapes are indistinguishable from wild-type FLNa (Figure S2A), and these mutations do not affect FLNa's actin gelation activity (data not shown).


Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

Nakamura F, Heikkinen O, Pentikäinen OT, Osborn TM, Kasza KE, Weitz DA, Kupiainen O, Permi P, Kilpeläinen I, Ylänne J, Hartwig JH, Stossel TP - PLoS ONE (2009)

FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP.(A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654154&req=5

pone-0004928-g002: FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP.(A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.
Mentions: FLAG-tagged full-length FLNa pulled down full-length FilGAP in vitro, and deletion of FLNa Ig repeat 23 abolished this interaction (Figure 2A), consistent with previous results [12]. The interaction was diminished or abolished respectively by deletion of hinge 2 and FLNa Ig repeat 24 (Figure 2A). Deletion of the hinge 2 from the C-terminal of IgFLNa23-24 disrupted FilGAP binding (Figure 2B). A hinge 2 plus IgFLNa24 construct does not bind FilGAP (data not shown), indicating that hinge 2 is not the FilGAP-binding site. Electron micrographs of mutant FLNa constructs demonstrate that their shapes are indistinguishable from wild-type FLNa (Figure S2A), and these mutations do not affect FLNa's actin gelation activity (data not shown).

Bottom Line: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction.FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations.Disease-related FLNa mutations have demonstrable effects on FLNa function.

View Article: PubMed Central - PubMed

Affiliation: Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. fnakamura@rics.bwh.harvard.edu

ABSTRACT

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans.

Methodology/principal findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress.

Conclusions/significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

Show MeSH
Related in: MedlinePlus