Fig 1.
Structure of the Skp1–FBG3 complex.
(A) Sequence conservation and secondary structure elements of Fbs family proteins. The alignment was generated by ClustalW [25]; α-helices are depicted as coils, β-strands as arrows, and turns by the letters TT. Conserved residues are boxed in white on a red background and similar residues are boxed in red on a white background. F-box and linker domains are boxed in cyan and purple, respectively. β2-β3, β5-β6, β7-β8, and β9-β10 loops are indicated by green boxes. Filled green circles indicate residues forming the carbohydrate-binding pocket. The figure was generated by ESPript [26]. (B) Overall structure of the complex. Skp1 and FBG3 are colored blue and red, respectively. The secondary structure elements for Skp1 and FBG3 are labeled in blue and red, respectively. Dotted lines represent disordered regions.(C) Model of the SCFFBG3 complex bound to E2. Cul1, Rbx1, Skp1, FBG3, and E2 are colored green, orange, blue, red, and yellow, respectively. A model of SCFFBG3 was simply constructed by superposition of the Skp1 subunits from the Skp1–FBG3, Skp1–Fbs1, and Skp1–Cul1–Rbx1 structures [27] (PDB ID code 1LDK), the RING-finger domains derived from Rbx1 and the c-Cbl subunit of the c-Cbl-UbcH7 structure [28] (PDB ID code 1FBV), and the E2 subunits of the c-Cbl-UbcH7 structure, using the program LSQKAB [29].
Table 1.
Data collection and refinement statistics.
Fig 2.
Comparison of the linker domains in Skp1–FBG3 and Skp1–Fbs1.
(A) Comparison of the crystal structure of the Skp1 (blue)–FBG3 (red) complex and the Skp1 (yellow)–Fbs1 (green) complex. (B) Magnified view of the linker region of the substrate-binding domain.
Fig 3.
Comparison of the substrate-binding domain (SBD) between FBG3 and Fbs1.
(A) Stereo view of the comparison between SBD of FBG3 (red) and SBD of Fbs1 (green). (B, C) Comparison of the intramolecular hydrogen bonds in the SBD of FBG3 (B) and SBD of Fbs1 (C). Hydrogen bonds are represented as dashed lines. The loops β2-β3, β5-β6, β7-β8, and β9-β10 are colored blue, cyan, magenta, and yellow, respectively. The residues of the hydrogen bonding pair are depicted as stick models. The carbohydrate-binding residues are depicted as line models. (D, E) The schematic view of the hydrogen bond networks between four loops in FBG3 (D) and Fbs1 (E). The loops β2-β3, β5-β6, β7-β8, and β9-β10 are labeled and colored as in (B, D). Hydrogen bonds are represented as solid lines. The residues of the hydrogen bonding pair are labeled beside each loop to which they belong.
Fig 4.
In vitro RNase B binding activities of the Fbs1 and its mutants using pull-down assay.
(A) Characterization of the crucial loops in Fbs1 for carbohydrate binding. (B) Comparison of relative RNase B binding activities between wild type (control) and loop mutants shown in (A). Three independent pull-down assays were analyzed. Error bars represent means ± S.E. (C) Determination of pivotal residues in the loop β5-β6 for the carbohydrate-binding pocket formation. (D) Effects of mutations introduced in non-conserved residues in the loop β5-β6 shown in (C). Three independent pull-down assays were analyzed. Error bars represent means ± S.E.
Fig 5.
In vitro RNase B binding activities of the Skp1–FBG3 complex and its mutants using pull-down assay.
(A) Determination of the crucial loops for the carbohydrate-binding pocket formation in FBG3. (B) Comparison of relative RNase B binding activities between Skp1–Fbs1 wild type (control) and Skp1–FBG3 loop mutants shown in (A). Three independent pull-down assays were analyzed. Error bars represent means ± S.E.