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Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain

Abstract

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine–actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.

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Figure 1: Structure of the FH2–actin complex.
Figure 2: Relationship between the bridge-bound actin dimer and that in the Holmes model of the actin filament.
Figure 3: The bridge blocks actin addition at the barbed end but not the pointed end.
Figure 4: Actin regulation by linked FH2 domain mutants.
Figure 5: Model for FH2-mediated barbed end dynamics.

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Acknowledgements

We thank S. Hill and C. Brautigam for technical assistance; Y. M. Chook, M. Kikkawa, R. Ranganathan and D. Morgan for discussion and critical reading of the manuscript; S. Padrick for assistance with modelling FH2-mediated polymerization and depolymerization; and P. Mishra for help with light scattering experiments. This work was supported by grants from the NIH to M.K.R. T.O. was supported by the Human Frontier Science Program. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the US Department of Energy, Office of Energy Research.

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Correspondence to Michael K. Rosen.

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Supplementary information

Supplementary Figures S1–S5

Five Supplementary Figures showing detailed analyses of the structure and supporting biochemical data. (DOC 1778 kb)

Supplementary Tables

Two Supplementary Tables summarizing actin regulatory properties of FH2 mutants and actin-actin interactions in the FH2-actin crystal. (DOC 24 kb)

Supplementary Discussion

Structural description and functional validation of the knob and post actin binding sites of the FH2 domain. (DOC 38 kb)

Supplementary Methods

Details of the experimental methods including protein preparation, X-ray crystallography and NMR spectroscopy. (DOC 56 kb)

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Otomo, T., Tomchick, D., Otomo, C. et al. Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain. Nature 433, 488–494 (2005). https://doi.org/10.1038/nature03251

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