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. 2013 Oct 18;8(10):e78907. doi: 10.1371/journal.pone.0078907

Figure 1. The F-boxes of SPBP and RAI1 interact with their atypical PHD fingers.

Figure 1

(A) Schematic illustration of domain structures of human SPBP (1960aa) and RAI1 (1906aa). TAD: trans-activation domain, NLS: nuclear localization signals, DBD: DNA-binding domain, ePHD/ADD: extended plant homeodomain, Q1/Q2: glutamine-rich stretches, grey boxes A to G: represents evolutionary conserved regions with strong homology between SPBP and RAI1. (B) A cartoon depicting a model of the ePHD domain of SPBP. In this model the F box interacts with the PHD domain of SPBP to form an ePHD/ADD domain containing two interacting zinc-finger modules. The interleaved zinc-ligation topology of the PHD domain is shown. Abbreviations: Zn, zinc; C, cysteine; H, histidine; N, N-terminal end; C, C-terminal end. (C) A schematic presentation of the ePHD/ADD domain of SPBP is shown. Asterisks mark Cys and His residues that may serve as zinc ligands. The F box is indicated in dark grey. The N- and C-terminal amino acid positions demarcating the SPBP ePHD/ADD domain are shown. Truncated versions of the ePHD/ADD domains of SPBP and RAI1, containing zinc-ligands 3-12 interact with their own and the others F box, as revealed by two-hybrid analysis. The F boxes of both proteins were fused to GAL4-AD and tested towards GAL4-DBD constructs of SPBP(ePHD), SPBP(ePHD3-12), RAI1(ePHD3-12), MLL1(ePHD), MLL2(ePHD) and MLL3(ePHD). Interactions are indicated by growth on QDO medium. Yeast cell suspensions were spotted onto plates and allowed to grow at 30°C for 3 days. (D) Deletion- and point-mutational analysis of the ePHD/ADD domain of SPBP using the two-hybrid interaction system. SPBP (ePHD/ADD) (black), SPBP (ePHD3-12) (grey) and the PHD finger, named SPBP (ePHD5-12) (white) were fused to GAL4-DBD and tested for interaction with the F box of SPBP fused to GAL4-AD. In addition, three constructs obtained by site-directed mutagenesis, generating mutations in the putative zinc-ligands 1 and 2 in the F box of SPBP(ePHD/ADD) were fused to GAL4-DBD, and tested for interaction to the SPBP F box fused to GAL4-AD. Cysteine(s) are mutated to alanine(s) as indicated in (C).