Fig. 6. The FH2 domain of FOZI-1 is unusual and does not affect actin dynamics. (A) Alignment of FH2^FOZI-1 with other FH2 domains, calculated with T-coffee version 2.03 (Notredame et al., 2000). FH2^FOZI-1 shows 9% to 20% sequence identity to other sequences in the alignment (49 FH2 domains from Pfam; only four shown here), which is in the range of sequence identities between the remaining FH2 domain sequences in the alignment. The sequence identities of FH2^FOZI-1 and FH2 domains of known structure, yeast FH2^Bni-1 [PDB-id:1y64; (Otomo et al., 2005)] and mouse FH2^Dia1 [PDB-id:1v9d; (Shimada et al., 2004)], are 16% and 14%, respectively. The profile-profile alignment program hmap (Tang et al., 2003) assigns a very good E-value (1.4e-26) for alignments between FH2^FOZI-1 and yeast FH2^Bni1. Secondary structure prediction for FH2^FOZI-1 also agrees well with the secondary structure found in the known structures (colored bar above alignment for FH2^Bni1). Neither Ile1431 nor Lys1601, which are crucial for the actin nucleation activity of the FH2 domains are conserved in FH2^FOZI-1 (red and black arrow). The colored bar at the bottom shows sequence conservation as calculated previously (Valdar, 2002) (red, high degree of conservation; blue, low degree of conservation). (B) Dimeric structure of the FH2 domain of yeast Bni1 (Otomo et al., 2005). (C) FH2^FOZI-1 does not stimulate actin polymerization. The FH2 domain of mouse Dia1 serves as a positive control. (D) FH2^FOZI-1 multimerizes. Isolated FH2^FOZI-1 (predicted to be 42 kDa) migrates as a single band (lane 1, -BMH). Treatment of FH2^FOZI-1 with the crosslinking reagent Bis-maleimidohexane (BMH) produces discrete slower migrating bands (lane 3, +BMH). The putative FH2 multimer is not present if the protein sample is denatured prior to crosslinking (lane 2, +SDS+BMH). FH2^FOZI-1 was detected by immunoblotting.