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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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Related in: MedlinePlus

Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction.(A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.
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pone-0004928-g004: Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction.(A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.

Mentions: To verify that the interaction site with FilGAP is on the CD face of IgFLNa23 we next mutated the hydrophopic M2474 to negatively charged glutamate. Indeed, the point mutation M2474E in IgFLNa23 abolished the interaction of full length FLNa with recombinant FilGAP C-terminal fragment and with full length FilGAP in vitro as predicted (Figure 4A). However, the mutant filamin fully retained F-actin gelation activity, and its morphology is indistinguishable from wild-type FLNa in electron micrographs (Figure S2). NMR spectra also showed that M2474E IgFLNa23 was fully folded (Figure S5). Thus, this mutation further confirms that also in the context of full length FilGAP and FLNa, the major FilGAP binding site is at the CD face of IgFLNa23.


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)

Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction.(A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004928-g004: Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction.(A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.
Mentions: To verify that the interaction site with FilGAP is on the CD face of IgFLNa23 we next mutated the hydrophopic M2474 to negatively charged glutamate. Indeed, the point mutation M2474E in IgFLNa23 abolished the interaction of full length FLNa with recombinant FilGAP C-terminal fragment and with full length FilGAP in vitro as predicted (Figure 4A). However, the mutant filamin fully retained F-actin gelation activity, and its morphology is indistinguishable from wild-type FLNa in electron micrographs (Figure S2). NMR spectra also showed that M2474E IgFLNa23 was fully folded (Figure S5). Thus, this mutation further confirms that also in the context of full length FilGAP and FLNa, the major FilGAP binding site is at the CD face of IgFLNa23.

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