The structure of the GPIb-filamin A complex

Abstract

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.

Figure 1.

Identification of protein domains responsible for GPIbα/FLNa interactions. (A) Amino acid sequence of the synthetic 22-mer peptide (GPIbα556-577) that binds FLNa and was used for crystallography. (B) Binding of purified recombinant human FLNa (2μg 400μL^-1) to GST-GPIbα515-610 (5μg 400μL^-1) is inhibited by the 22-mer peptide, GPIbα556-577, in a dose-dependent manner. (C) Full-length FLNa (2μg 400μL^-1) binds the GPIbα556-577 peptide immobilized on Sepharose 4B but not Sepharose 4B alone. (D) In vitro binding of GST-IgFLNa17, -18, and -19 (2 μg each) to the GPIbα556-577 peptide beads. Top and bottom rows indicate bound and input proteins to GPIbα556-577, respectively. (E) Tag-free IgFLNa17 binds GPIbα556-577 peptide with higher affinity than IgFLNa19. Increasing amount of purified IgFLNa17 or -19 were incubated with GPIbα556-577 peptide beads. (F) IgFLNa17 is the major binding site for GPIbα556-577 peptide. Various amounts of IgFLNa17 and 19 at 1:1 molar ratio were incubated with GPIbα556-577 peptide beads in the pull-down experiment shown. (G) FLNa17 but not FLNa19 interferes the binding of full-length FLNa to GPIbα peptide beads. Various amounts of FLNa17 or -19 were mixed with 1 μg purified full-length FLNa and then incubated with GPIbα556-577 peptide beads. (B-G) Bound proteins were resolved by SDS-PAGE, and stained with Coomassie blue.

Figure 2.

Structure of IgFLNa17 alone and in complex with the GPIbα peptide. (A) A family of 20 NMR structures of the IgFLNa17 in solution. (B) Asymmetric unit of the crystal. Two molecules of IgFLNa17 are colored green and blue (chains A and B, respectively). The GPIbα peptides are magenta (chain S) and gold (chain T). The N and C termini of all chains are shown. Note that the peptide T interacts with both FLN molecules. (C) Superimposition of the crystal structure (green) and the mean NMR structure (red). (D) Surface properties of the FLNa-GPIbα556-577 interaction. The accessible surface of IgFLNa17 has been colored according to surface potential and the peptide is shown on the top (gold and atom color). This representation shows the extended conformation of the peptide in the complex and the hydrophopic character of the interaction. (E) Main chain hydrogen bonding between the peptide (green) and IgFLNa17 strand C. (F) Details of the peptide interaction. An electron density map calculated with the final model without the peptide at the σ level of 1.4 shows that the side chains of the peptide are well defined. The G1897 and C1912 residues of FLNa that were mutated in subsequent studies are shown in magenta. Residue numbers for the peptide are shown in black and for FLNa in green.

Figure 3.

Obliteration of the binding of full-length FLNa to the GPIb complex in CHO cells by the point mutations in IgFLNa17 and by thiol alkylation. (A) ClustalW alignment of domain 17 of FLN isoforms. Residues inferred from the atomic structure to be involved in interactions with GPIbα and tested by mutagenesis are indicated by the gray bars. Potential targets of NEM are indicated by the black bars. (B) Double mutations at G1897D and C1912D of IgFLNa17 abolish IgFLNa17/GPIb binding. Increasing amounts of purified MBP-IgFLNa17 (WT, wild type) and its mutants (G1897D, I1910M, C1912D, and G1897D+C1912D) were incubated with GPIbα556-577–peptide beads, and bound proteins were detected by SDS-PAGE followed by Coomassie blue staining. (C) GFP-FLNa and its mutants were expressed in CHO-GPIb/IX cells and coimmunoprecipitated with the GPIb-IX complex. GFP-FLNa is detected by immunoblot with anti-GFP mAb. The intensity of the band was analyzed by densitometry and the relative intensity graphed. Data are presented as mean ± SD of 3 independent experiments. (D) FLN is expressed in CHO-GPIb/IX cells and coimmunoprecipitated with the GPIb-IX complex. The linkage is uncoupled by treating the cell lysate with cystein-blocking reagent, NEM. Since only anti-FLNb, but neither anti-FLNa nor anti-FLNc antibodies available cross-react with hamster FLN, immunoblot was performed with anti-FLNb mAb (1-11c). IP indicates immunoprecipitation; IB, immunoblot.

Figure 4.

Schematic drawing and electron micrograph of the GPIb-FLNa complex from human platelets. (A) The cytoplasmic domain of GPIbα of the GPIb-IX-V complex binds to actin filaments through FLNa. (B) GPIb-FLNa complex from human platelets. Coomassie-blue-stained SDS-PAGE (10%) of a concentrated GPIb-FLNa complex (left). Immunoblot of the GPIb-FLN complex with anti-GPIbα mAb (WM23) (center). The same blotting membrane was subsequently blotted with an anti-FLNa mAb 3-14 (right). (C) Electron micrographs of rotary-shadowed recombinant FLNa molecules (top) and GPIb-FLNa complexes (bottom).

Figure 5.

ClustalW alignment of GPIbα, integrin, and dopamine receptor cytoplasmic tail sequences. The FLNa-binding site of GPIbα and integrin β7 from the crystal structures and the corresponding sites of other integrins and dopamine D2 and D3 receptors are boxed. Potential phosphorylation sites (Tyr, red; Thr and Ser, blue) of integrins are indicated. A putative PKC phosphorylation site in D2 and D3 receptors are marked in yellow.