Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206

Abstract

Zfp206 (also named as Zscan10) belongs to the subfamily of C(2)H(2) zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 Å crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C(2)H(2) transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development.

Figure 1.

Overall structure of Zfp206SCAN. (A) The Zfp206SCAN domain-swapped dimer is formed by packing helix H2 of one monomer (green) against helices H3 and helix H5 of the opposing monomer (orange). (B) The (2F_O-F_C) map of the dimer interface involving residues Arg13 and Arg31 in molecule 1 and Gln56 and Glu50 (as indicated in Figure 2A) in molecule 2 of Zfp206SCAN contacted by hydrogen bond. The electron density (2F_o-F_c) is displayed at the 0.5σ level. (C) The (2F_O-F_C) map of the dimer interface involving residues Ile48 and Leu49 in molecule 1 and Cys34, Ile48 and Leu49 of molecule 2 (as indicated in Figure 2A).

Figure 2.

In vitro MBP pull-down assay of Zfp206SCAN and other SCAN domains. (A) Multiple sequence alignments of the SCAN domains used in MBP pull-down assay. Secondary structure elements of SCAN domains are shown under the alignments as orange blocks. Arrows are for polar interface residues, and dots are for hydrophobic residues seen in the structure of the Zfp206SCAN homodimer. The numbers are shown above the alignment mark residues with the conserved portion of the SCAN domain for easier comparison. The red arrows and dots are the mutated residues described in the later study (Figure 4). (B) Pull-down of non-tagged Zfp206SCAN using amylose beads pre-coated with recombinant MBP or MBP-SCAN fusion proteins. The prey proteins were detected by SDS–PAGE and Coomassie stain, and identified to be Zfp206SCAN by mass spectrometry. The upper bands in lanes 9 and 10 are the dimer of MBP167SCAN. The additional bands seen in lanes 14, 15, 32, 39 and 43 are non-specific proteins produced during purification.

Figure 3.

Model for Zfp206SCAN-Zfp110SCAN heterodimer. (A) Model for Zfp206SCAN-Zfp110SCAN created by superimposing Zfp110SCAN monomer on Zfp206SCAN molecule of the homodimer followed by MD simulations. In the model, the Zfp206SCAN monomer is presented in red and the Zfp110SCAN monomer is presented in blue. (B) Based on the model of Zfp206SCAN and Zfp110SCAN heterodimer, interface residues (shown in green) including Arg 13, Arg 31, Ile 48 and Leu 49 in Zfp206SCAN monomer and Cys 34, Leu 38 and Leu 49 in Zfp110SCAN were chosen for mutations to disrupt interactions between the two monomers. (C–D) Homodimerization of Zfp206SCAN and Zfp110SCAN. (C) Fifteen percent SDS–PAGE showing purified Zfp206 in lane 2 and Zfp110 in lane 3 and molecular weight standards in lane 1 in kilo Daltons. (D) Size-exclusion chromatogram showing that the Zfp206SCAN as well as the Zfp110SCAN elute as a single symmetric peaks corresponding to the molecular weight of the homodimeric forms of the proteins (21 and 24.8 kDa). The cartoons represent the protein conformations.

Figure 4.

Role of the amino-terminal helix 1 for Zfp206SCAN and Zfp110SCAN heterodimerization and mutations based on the Zfp206SCAN-Zfp110SCAN heterodimer. (A) The intramolecular linker-mediated Zfp206-Zfp110 heterodimer elutes at the volume corresponding to the theoretical molecular weight of a monomeric fusion protein 23 KDa, which is virtually identical with the elution profile of un-connected Zfp206SCAN homodimer (21 KDa). Oppositely, the Zfp206-ZNF174 elutes at the volume corresponding to a higher molecular weight indicating the formation of an intermolecular homodimer. The profile of a molecular weight standard is overlaid as green curve. (B) 15% SDS–PAGE gel shows purified Zfp206-ZNF174 (lane 2) and Zfp206-Zfp110 (lane 4) and molecular standards (lane 1 and 3; weights in kilo Daltons). (C) Elution profile of the linker mediated intramolecular Zfp206-Zfp110 heterodimer (left) and SDS–PAGE of the eluted fractions (right). (D) Elution profile of the Zfp206-/Zfp110_H1¹⁷⁴ protein suggesting the formation of an intermolecular homodimer instead of the linker-mediated heterodimer seen in (C). Cartoon drawing of the various complexes are shown as inset of the chromatograms with the Zfp206SCAN in red, the Zfp110SCAN in blue and the ZNF174SCAN in green. The linker is illustrated with a black line and the helix1 of the ZNF174SCAN that was introduced into the Zfp110SCAN is shown as green bar. (E) Zfp206-4mut-Zfp110 intermolecular homodimer (Zfp206SCANI48AL49AR13AR31A-Zfp110SCAN) and (F) Zfp206-Zfp110-3mut intermolecular homodimer (Zfp206SCAN-Zfp110SCANC36AL40AL51A).

Figure 5.

Zfp206 interacts with Zfp110 in vivo. (A) Anti-Zfp110 antibody was used to immunoprecipitate (IP) Zfp110 and the co-IP products were analyzed by western blot (WB) with an anti-Zfp206 antibody to test whether Zfp110 interact with Zfp206. Vice versa, (B) anti-Zfp206 antibody was used to immunoprecipitate Zfp206, and co-IP products were analyzed by WB with an anti-Zfp110 antibody to check whether Zfp206 interacts with Zfp110.

Figure 6.

Structural alignment of Zfp206SCAN homodimer with ZNF174SCAN homodimer (PDB entry 1Y7Q) and MZF1SCAN homodimer (PDB entry 2FI2). The Zfp206SCAN is shown in green; the ZNF174SCAN is shown in pink and MZF1SCAN is shown in yellow.