Limits...
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.

Show MeSH

Related in: MedlinePlus

Localization of FLNa-FilGAP binding site.(A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654154&req=5

pone-0004928-g001: Localization of FLNa-FilGAP binding site.(A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.

Mentions: Most of the protein constructs used for in vitro binding assay were obtained in good yield and purity (Figure S1A). Figure 1A shows a schematic diagram of FilGAP structure and demonstrates that the C-terminal 100 residues (649–748 amino acid, aa) of FilGAP tagged to a glutathione S-transferase-hexahistidine (GST-His) interact with purified full-length FLNa in vitro, consistent with previous results [12]. Further deletion constructs identified that the last 32 residues (717–748aa, FilGAPC32), but not residues 649–729 of the predicted coiled-coil domain by EMBnet COILS, are the FLNa-binding site (Figure 1A). FilGAPC32 tagged to a maltose binding protein followed by hexahistidine (MBP-His), however, did not pull down with FLAG-FLNa (Figure 1B), and a synthetic peptide of FilGAPC32 immobilized on Sepharose also did not precipitate FLNa (data not shown). Analytical gel filtration demonstrated that full-length FilGAP is a dimer and FilGAP lacking residues 649–725, which contains the minimum FLNa-binding site (residues 726–734, see below), is a monomer (Figure 1D and Figure S1B) and does not form a tight complex with FLNa (Figure 1C). MBP-His-FilGAP constructs containing residues 649–729 eluted as a decamer from gel filtration columns, whereas MBP-His-FilGAPC32 was monomeric (Figure 1D and Figure S1B).


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)

Localization of FLNa-FilGAP binding site.(A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654154&req=5

pone-0004928-g001: Localization of FLNa-FilGAP binding site.(A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.
Mentions: Most of the protein constructs used for in vitro binding assay were obtained in good yield and purity (Figure S1A). Figure 1A shows a schematic diagram of FilGAP structure and demonstrates that the C-terminal 100 residues (649–748 amino acid, aa) of FilGAP tagged to a glutathione S-transferase-hexahistidine (GST-His) interact with purified full-length FLNa in vitro, consistent with previous results [12]. Further deletion constructs identified that the last 32 residues (717–748aa, FilGAPC32), but not residues 649–729 of the predicted coiled-coil domain by EMBnet COILS, are the FLNa-binding site (Figure 1A). FilGAPC32 tagged to a maltose binding protein followed by hexahistidine (MBP-His), however, did not pull down with FLAG-FLNa (Figure 1B), and a synthetic peptide of FilGAPC32 immobilized on Sepharose also did not precipitate FLNa (data not shown). Analytical gel filtration demonstrated that full-length FilGAP is a dimer and FilGAP lacking residues 649–725, which contains the minimum FLNa-binding site (residues 726–734, see below), is a monomer (Figure 1D and Figure S1B) and does not form a tight complex with FLNa (Figure 1C). MBP-His-FilGAP constructs containing residues 649–729 eluted as a decamer from gel filtration columns, whereas MBP-His-FilGAPC32 was monomeric (Figure 1D and Figure S1B).

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