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FMNL3 FH2–actin structure gives insight into formin-mediated actin nucleation and elongation

Abstract

Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

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Figure 1: Structure of FMNL3 FH2 domain bound to TMR-actin.
Figure 2: Distinct actin-actin interactions in the FMNL3 FH2–actin complex compared to the Bni1p FH2-actin complex.
Figure 3: FH2-actin binding interfaces.
Figure 4: Effects of FMNL3 mutations on actin filament elongation.
Figure 5: Effects of FMNL3 mutants on formation of filopodia in Jurkat cells.
Figure 6: Model for FMNL3-mediated actin assembly.

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Acknowledgements

We thank our undergraduates A. Kelley for mutagenesis and cloning and M. Lee for crystallization trials and screening; J. Moseley for thoughtful comments and careful reading of the manuscript; and S. Corcoran and the staff at the GM/CA CAT 23-ID-D beamline at Argonne National Laboratories Advanced Photon Source (APS) Source; and C. Bahl and D. Madden for help with the structural determination and characterization. The human α1β2 expression vector was a gift from J. Cooper (Washington University, St. Louis, Missouri, USA). This work was supported by US National Institutes of Health grants R01 GM069818 (to H.N.H.) and F31 GM089149 (to E.G.H.) as well as Howard Hughes Medical Institute predoctoral fellowship 52006921 and National Science Foundation GK-12 fellowship 0947790 (to M.E.T.). GM/CA at APS has been funded in whole or in part with US federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (Y1-GM-1104). Use of the APS was supported by the US Department of Energy, Basic Energy Sciences, Office of Science (contract DE-AC02-06CH11357).

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M.E.T. purified, crystallized and characterized the FH2–actin complex and also collected data on the crystals and determined the structure. E.G.H. performed the pyrene-actin assays. T.J.G. carried out the cellular characterization and analysis of the mutant protein constructs. The experimental design and data analysis was carried out by M.E.T., H.N.H. and F.J.K. The manuscript was prepared by M.E.T., H.N.H. and F.J.K.

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Correspondence to Henry N Higgs or F Jon Kull.

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Thompson, M., Heimsath, E., Gauvin, T. et al. FMNL3 FH2–actin structure gives insight into formin-mediated actin nucleation and elongation. Nat Struct Mol Biol 20, 111–118 (2013). https://doi.org/10.1038/nsmb.2462

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