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Structural mechanism of integrin inactivation by filamin

Nature Structural & Molecular Biology volume 22, pages 383–389 (2015)Cite this article

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Abstract

Activation of heterodimeric (αβ) integrin is crucial for regulating cell adhesion. Binding of talin to the cytoplasmic face of integrin activates the receptor, but how integrin is maintained in a resting state to counterbalance its activation has remained obscure. Here, we report the structure of the cytoplasmic domain of human integrin αIIbβ3 bound to its inhibitor, the immunoglobin repeat 21 of filamin A (FLNa-Ig21). The structure reveals an unexpected ternary complex in which FLNa-Ig21 not only binds to the C terminus of the integrin β3 cytoplasmic tail (CT), as previously predicted, but also engages N-terminal helices of αIIb and β3 CTs to stabilize an inter-CT clasp that helps restrain the integrin in a resting state. Combined with functional data, the structure reveals a new mechanism of filamin-mediated retention of inactive integrin, suggesting a new framework for understanding regulation of integrin activation and adhesion.

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Figure 1: Interaction of FLNa-Ig21 with αIIbβ3 CTs.
Figure 2: FLNa-Ig21 promotes ternary-complex formation with αIIb CT and β3 CT.
Figure 3: Structure of the FLNa-Ig21–αIIb CT–β3 CT ternary complex.
Figure 4: A topology of FLNa-Ig21 bound to the αIIbβ3 CT heterodimer at the inner-membrane surface.
Figure 5: Functional evidence of FLNa-Ig21–mediated inhibition on integrin activation.
Figure 6: Comparison of FLNa-Ig21–bound integrin αIIbβ3 CT complexes (a) with talin-F3–bound β1 CT (b) (derived from PDB 3G9W), showing how FLNa-Ig21 may compete with both β3-MP and β3-C for binding to talin-F3.

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Acknowledgements

We wish to thank X. Zhang, Y.-Q. Ma, J.-H. Ma and S. Misra for useful discussions and technical assistance. This work was supported by US National Institutes of Health grants to J.Q. (GM062823), V.P.Y. (DK102020) and E.F.P. (HL073311).

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  1. Jianmin Liu and Mitali Das: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA

    Jianmin Liu, Mitali Das, Jun Yang, Sujay Subbayya Ithychanda, Valentin P Yakubenko, Edward F Plow & Jun Qin

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Contributions

J.L. and J.Q. conceived the study. J.L. performed all NMR and biochemical studies with the assistance of J.Y. and S.S.I. M.D. performed all functional experiments. All authors were involved in data interpretation and discussion. J.L. and J.Q. wrote the manuscript with contributions from all other authors.

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Correspondence to Edward F Plow or Jun Qin.

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Integrated supplementary information

Supplementary Figure 1 Interaction of FLNa-Ig21 with αIIbβ3 CTs.

(a) HSQC of 0.1mM 15N-labeled β3-N in the absence (black) and presence (red) of 0.1mM FLNa-Ig21, pH 6.4, 25°C. (b) Representative real-time sensorgrams of the binding between β3-N and FLNa-Ig21 by SPR analysis (n=2). The real-time binding curves were fitted using a global fitting algorithm to a 1:1 binding model, resulting in the KD~223μM. (c) 2D NOESY fingerprint region of β3-N amides showing sequential NHi-NHi+1 NOEs in the presence of GST-FLNa-Ig21. Note that β3-CT has no binding to GST only (data not shown). (d) Comparison of representative HSQC regions between 15N-labeled FLNa-Ig21 (black) and 15N-labeled FLNa-Ig21-β3-C chimera (red) showing dramatic chemical shift difference consistent with the strong β3-CT binding to FLNa-Ig21 in the chimera. The dramatic spectral perturbation pattern (see the representative peaks) is similar to the slow-exchange FLNa-Ig21 interaction with other strong ligands such as integrin β7-C, migfilin, and GP1bα12. (e). 0.1mM 15N-labeled FLNa-Ig21-β3-C chimera in the absence (black) and presence of 0.2mM β3-N showing significant perturbation of selective residues. (f). 2D NOESY fingerprint region of αIIb-CT showing sequential NHi-NHi+1 NOEs in the presence of GST-FLNa-Ig21. All spectra were performed at 25°C, pH 6.4. (g). 2D NOESY fingerprint region of αIIb-CT showing K989-F992 Hαi-NHi+3 and Hαi-Hβi+3 NOEs in the presence of GST-FLNa-Ig21.

Supplementary Figure 2 FLNa-Ig21 promotes ternary-complex formation with αIIb CT and β3 CT.

(a) Representative real-time sensorgrams of the binding between αIIb-CT and β3-CT by SPR analysis (n=2). The real-time binding curves were fitted using a global fitting algorithm to a 1:1 binding model, resulting in the KD~368μM. (b) Chemical shift perturbation profiles of 0.1mM 15N-labeled FLNa-Ig21 by 0.03mM β3-CT (top panel) and 0.03mM β3-CT L717K/L718K mutant (bottom panel) showing that the mutations did not affect the FLNa-Ig21/β3-CT interaction. Note that lower amount of WT β3-CT had to be used to reduce the line-broadening problem and perform the chemical shift mapping as compared to that of the β3-CT mutant. (c) HSQC of 0.1mM 15N-labeled 0.1mM β3-CT L717K/L718K mutant in the absence (black) and presence (red) of 0.1mM FLNa-Ig21 showing substantially reduced line-broadening problem as compared to Fig. 1A (upper panel), which allowed the chemical shift mapping of both perturbed β3-MP and β3-C regions (lower panel). (d) HSQC of 0.1mM 15N-labeled β3-CT L717K/L718K mutant in the absence (black) and presence (red) of 0.1mM FLNa-Ig21 and presence of 0.1mM FLNa-Ig21+0.2mM αIIb-CT (green) showing that αIIb-CT induced further chemical shift changes of β3-CT bound to FLNa-Ig21. (e) Representative intermolecular NOE strips: left panel, NOEs between FLNa-Ig21 and αIIb-CT; right panel, NOEs between between FLNa-Ig21 and β3-CT.

Supplementary Figure 3 Critical side chain interface of FLNa-Ig21–αIIb CT–β3 CT complex.

Overlay of 20 lowest energy structures showing the side chain convergence of critical interface residues as shown in Fig 3.

Supplementary Figure 4 Mutation effects of FLNa-Ig21–αIIb CT–β3 CT ternary complex.

(a) Chemical shift perturbation profiles of αIIb-CT in the presence of FLNa-Ig21 (upper panel) vs FLNa-Ig21 E2276A (lower panel) showing that the E2276A mutation impaired the FLNa-Ig21/αIIb-CT interaction. (b) Chemical shift perturbation profiles of β3-CT L717K/L718K in the presence of FLNa-Ig21 (upper panel) vs FLNa-Ig21 E2276A (lower panel) showing that the E2276A mutation has little effect on the FLNa-Ig21/β3-CT interaction. (c) HSQC of 0.1 mM 15N-labeled FLNa-Ig21 in the absence (black) and presence (red) of 0.2mM αIIb CT K994E/R997E mutant showing that the mutation diminished the FLNa-Ig21/αIIb CT interaction. (d) Transferred NOEs in the selected region of NOESY for 2mM aIIb K994E/R997E mutant in the absence (no NOEs) and presence (black) of 0.1mM MBP-β3 CT showing the αIIb/β3 CT interaction is preserved as shown for the WT αIIb CT previously13. (e) Chemical shift perturbation profile of 0.1mM 15N-labeled FLNa-Ig21 in the presence of β3-N (upper panel) vs FLNa-Ig21 A2268K in the presence of β3-N (lower panel) showing that the A2268K mutation impaired the FLNa-Ig21/β3-N interaction. (f). Chemical shift perturbation profile of 0.1mM 15N-labeled FLNa-Ig21 in the presence of 0.2mM αIIb-CT (upper panel) vs FLNa-Ig21 A2268K in the presence of αIIb-CT (lower panel) showing that the A2268K mutation has little effect on the FLNa-Ig21/αIIb-CT interaction. (g). FLNa-Ig21 expression is similar to F21EA and F21AK mutants (no statistically significant difference). Data represent 3 independent experiments and error bars indicate Mean±S.E.M as explained in the legend for Fig 5.

Supplementary Figure 5 Interaction of FLNa repeats with αIIb CT and β3-N.

(a) 0.1mM 15N-labeled FLNa-Ig9 with 0.5mM αIIb CT and (b) with 0.5mM β3-N. (c) 0.1mM 15N-labeled FLNa-Ig12 with 0.2mM αIIb CT and (d) with 0.2mM β3-N. (e). 0.1mM 15N-labeled FLNa-Ig17 with 0.2mM αIIb CT and (f) with 0.2mM β3-N. (g) 0.1mM 15N-labeled FLNa-Ig19 with 0.2mM αIIb CT and (h) with 0.2mM β3-N. All spectra show the interaction as judged by the selective chemical shift changes. Note that higher ratio of αIIb CT or β3-N was used due to their weaker interaction with FLNa-Ig9.

Supplementary Figure 6 Comparison of several αIIbβ3 CT complex structures.

(a). Structure of αIIbβ3 TMCD determined in bicelles17. The dotted box indicates the membrane-proximal clasp that was shown to be embedded in the membrane17, which is inaccessible to cytosolic binding proteins. The region in red highlights the reverse turn conformation. This red region corresponds to the red region in (b)-(d). (b) αIIbβ3 CT complex bound to FLNa-Ig21 with αIIb membrane-proximal region being helical. (c) Overlay of the membrane-proximal clasp between the structure in (b) and a previous structure (PDB 1M8O13). (d) Overlay of the membrane-proximal clasp between the structure in (b) and another previous structure (PDB 2KNC19).

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Liu, J., Das, M., Yang, J. et al. Structural mechanism of integrin inactivation by filamin. Nat Struct Mol Biol 22, 383–389 (2015). https://doi.org/10.1038/nsmb.2999

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