Figure 3. Recognition of TRF1 by Fbx4.

(A) An overall view of the Fbx4_G-TRF1_TRFH interaction. The interacting structural elements of Fbx4_G (β6 and αD) and TRF1_TRFH (βA, α2 and α3) are colored in cyan and orange, respectively, and the rest of Fbx4_G and TRF1_TRFH in blue and yellow.

(B) Stereo view of the Fbx4_G-TRF1_TRFH interface. Fbx4_G and TRF1_TRFH interacting residues are presented as stick models. The Fbx4_G-TRF1_TRFH intermolecular hydrogen bonds are shown as dashed magenta lines.

(C) The αD helix of Fbx4_G sits on a hydrophobic surface formed by μA and helices of α2 and α3 of TRF1_TRFH.

(D) Fbx4_G binding is TRF1_TRFH specific. Loop L₂₃ of TRF2_TRFH (green) adopts a different conformation than that of TRF1_TRFH (yellow) so that it cannot form an intermolecular β-sheet interaction with Fbx4_G. The surface representation shows that the side chain of TRF2 Leu93 collides with μ6 of Fbx4_G.

(E) Effects of the Fbx4 and TRF1 mutations on the Fbx4-TRF1 interaction in a yeast two-hybrid assay. Interaction of LexA-TRF1 with GAD-Fbx4 was measured as β-galactosidase activity. Data are average of three independent β-galactosidase measurements normalized to the wild-type Fbx4-TRF1 interaction, arbitrarily set to 100. Error bars in the graph represent standard deviation.

(F) GST pull-down assay of the wild-type and mutant Fbx4_G-TRF1_TRFH interactions.