Crystal structure of human DAAM1 formin homology 2 domain

Abstract

Posttranslational modification by ubiquitin regulates a broad range of cellular activities such as protein degradation, transcriptional regulation, endocytosis, and DNA repair. p62 is one of the proteins which is known to recognize poly-linked ubiquitin chains through the ubiquitin associated (UBA) domain. Physiological function of p62 is implicated in the formation of protein inclusions which can be observed in neurodegenerative diseases such as Huntington's disease. p62 is known to accumulate in ubiquitin-positive inclusions of polyubiquitinated proteins, and p62 protein lacking the UBA domain failed to form inclusions in HEK cells, suggesting the important role of the p62 UBA domain in the formation of those inclusions. We solved the crystal structure of the p62 UBA domain at 1.4 angstrom resolution. Phases were obtained by MAD technique using the selenomethionine derivative of the p62 UBA domain. The structure of the p62 UBA domain was a homodimer in a crystallographic asymmetric unit. Two molecules form a dimer with a large hydrophobic interface. Results of analytical ultracentrifugation and NMR spectroscopy strongly supported that the p62 UBA domain also adopts a dimer configuration under an aqueous condition. We further examining how this dimeric structure changes upon ubiquitin binding and whether the dimerization affects the binding to the specific polyubiquitin chains. 1 The University of Tokyo, Medical Genome Sciences, 1-1-1 Yayoi, B u n k y o -k u , To k y o , 11 3 -0 0 3 2 , J a p a n , 2 K y o t o U n i v e r s i t y, 5 4 Shogoinkawara-machi, Sakyo-ku, Kyoto 606-8507, Japan, 3 Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan, 4 Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe-shi, Hyogo 650-0017, Japan, E-mail kk087351@mgs. k.u-tokyo.ac.jp Reorganization of actin filaments is an essential process for cell motility, cell-cell attachment, and intracellular transport. Formin protein family promotes nucleation and elongation of the actin filament, which is catalyzed by the conserved Formin-homology 2 (FH2) domain. FH2 forms a dimer and directly binds to the barbed end of the actin filament. The active dimeric FH2 structure has been reported in yeast formin, Bni1p, but not in any mammalian formin. Dishevelled-associated activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. To elucidate the mechanism of the actin filament assembly by mammalian FH2, we crystallized human DAAM1 FH2. The native crystal belongs to the triclinic space group P1, with unit-cell parameters a = 69.2 Å, b = 91.9 Å, c = 97.7 Å, α = 98.1 , β = 90.3 , γ = 104.8 , and diffracts to 2.8 Å resolution. The structure was solved by multiple-wavelength anomalous dispersion method using the SeMet-labeled crystal, and refined to an Rfree value of 28.9% at 2.8 Å resolution. The present DAAM1 FH2 structure consists of five subdomains (termed as "lasso", "linker", "knob", "coiled-coil", and "post"), and forms a dimeric ring in a head-to-tail manner si...

Structural and functional insights into the Spir/formin actin nucleator complex

“…C-terminal Fmn-2 sequences (accession number Q9NZ56) encompassing the FH2 domain and FSI sequence motif have been modeled with the HHpred server. The resulting structural model of the Fmn-2 C-terminus superimposed perfectly with the crystal structure of the Daam1 FH2 dimer (PDB ID: 2Z6E) (Yamashita et al, 2007) and the Fmn-2-FSI crystal structure (PDB ID: 2YLE, 3R7G) (Vizcarra et al, 2011;Zeth et al, 2011) at the very C-terminal end. This enabled us to include the Spir-1-KIND structural data from the KIND/FSI complex (PDB ID: 2YLE) into our model structure.…”

MIM regulates vertebrate neural tube closure

“…1C). Recent structural studies have shown that the conformation of the Daam1 FH2 domain is stabilized by the -sheet-like structure within this coil-coiled region, suggesting a regulatory role for this linker region (Lu et al, 2007;Yamashita et al, 2007). Molecules that bind this FH2 linker region might regulate the ability of the FH2 domain of Daam1 to mediate actin nucleation.…”

Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing

“…This isoleucine is present in all formins examined in yeast and metazoans ( Ref. 1 and further unpublished sequence analysis), and the side chain protrudes toward the center of the FH2 donut from an ␣ helix in the knob, according to crystal structures of Bni1p, DAAM1, mDia1, and FMNL3 (14,30,39,44). 3 In the Bni1p/actin co-crystal (44), the isoleucine side chain engages in interactions with residues at the "barbed end cleft" of actin between subdomains 1 and 3, a similar position to that occupied by WH2 domains (45).…”