Crystal structure of human LDB1 in complex with SSBP2

Abstract

The Lim domain binding proteins (LDB1 and LDB2 in human and Chip in Drosophila) play critical roles in cell fate decisions through partnership with multiple Lim-homeobox and Lim-only proteins in diverse developmental systems including cardiogenesis, neurogenesis, and hematopoiesis. In mammalian erythroid cells, LDB1 dimerization supports long-range connections between enhancers and genes involved in erythropoiesis, including the β-globin genes. Single-stranded DNA binding proteins (SSBPs) interact specifically with the LDB/Chip conserved domain (LCCD) of LDB proteins and stabilize LDBs by preventing their proteasomal degradation, thus promoting their functions in gene regulation. The structural basis for LDB1 self-interaction and interface with SSBPs is unclear. Here we report a crystal structure of the human LDB1/SSBP2 complex at 2.8-Å resolution. The LDB1 dimerization domain (DD) contains an N-terminal nuclear transport factor 2 (NTF2)-like subdomain and a small helix 4-helix 5 subdomain, which together form the LDB1 dimerization interface. The 2 LCCDs in the symmetric LDB1 dimer flank the core DDs, with each LCCD forming extensive interactions with an SSBP2 dimer. The conserved linker between LDB1 DD and LCCD covers a potential ligand-binding pocket of the LDB1 NTF2-like subdomain and may serve as a regulatory site for LDB1 structure and function. Our structural and biochemical data provide a much-anticipated structural basis for understanding how LDB1 and the LDB1/SSBP interactions form the structural core of diverse complexes mediating cell choice decisions and long-range enhancer-promoter interactions.

Keywords: LIM domain binding protein 1 (LDB1); Wnt enhanceosome; crystal structure; dimerization; single-stranded DNA binding protein 2 (SSBP2).

Fig. 1.

Biochemical characterization of the LDB1/SSBP2 interaction. (A) Schematic diagram of human LDB1 and SSBP2 domain organization. The dimerization domain (DD), LDB1/Chip conserved domain (LCCD), nuclear localization sequence (NLS), and LIM interaction domain (LID) are indicated for human LDB1. The LUFS domain (residues 10 to 77) is indicated for human SSBP2. We determined the crystal structure of the LDB1(56–285)/SSBP2(1–94) complex. (B) LDB1 dimer binds to SSBP2 tightly, as shown by MBP pull-down assays. MBP-UL37 was used as a negative control. (C) SSBP2 LUFS domain is sufficient for its interaction with LDB1 as shown by MBP pull-down assays. (D) Binding affinities of 4 SSBP2 fragments with LDB1 were measured by SPR assays. Values are presented ± SD. (E) LDB1 LCCD is sufficient for its binding with SSBP2 as shown by MBP pull-down assays. (F) Binding-affinities of SSBP2 with 2 LDB1 fragments were measured by SPR assays.

Fig. 2.

Overall structure of the human LDB1/SSBP2 complex. (A) Structure of 1 LDB1 in complex with 1 SSBP2 dimer. One SSBP2 dimer binds to 1 LDB1 LCCD domain. LDB1 NTF2-like subdomain, H4-H5 subdomain, and LCCD are colored in orange, magenta, and red, respectively. SSBP2 dimers are colored in green and cyan. (B) The “LDB1-only” structure in the complex. There is an H4-H5 subdomain between the main dimerization domain (NTF2-like subdomain) and LCCD. (C) Two orthogonal views of the LDB1/SSBP2 complex structure, which contains a symmetric LDB1 dimer, with each LDB1 monomer interacting with 1 SSBP dimer.

Fig. 3.

Structure and mutagenesis analysis of the LDB1 dimerization interface. (A) Close-up view of the LDB1 dimerization interface of the NTF2-like subdomain and the H4-H5 subdomain. Residues examined in mutagenesis analysis are labeled in red. (B) SEC-MALS analysis of MBP-tagged wild-type LDB1(56–287) (WT), LDB1(56–287) mut1 (Y69D/F72D/R210A), and LDB1(56–287) mut2 (replacing H4-H5 with a short linker) in complex with SSBP2. Molecular weights measured by SEC-MALS are shown. (C) Alignment of SEC profiles of MBP-tagged LDB1 WT (in red), mut1 (in green), and mut2 (in blue) in complexes with SSBP2. Calculated molecular weights for WT MBP-tagged LDB1 dimer in complex with 2 SSBP2 dimers and MBP-tagged LDB1 mut1 and mut2 monomers in complex with 1 SSBP2 dimer are labeled.

Fig. 4.

The LDB1/SSBP2 interface and mutagenesis analysis of the LDB1/SSBP2 interface. (A) Close-up view of the LDB1 LCCD/SSBP2 interface. Residues examined in mutagenesis analysis are labeled in red. (B) Close-up view of the LDB1-DD/SSBP2 interface. (C) Close-up view of the interface between LDB1-LCCD and SSBP2 residues 77 to 94 that is C-terminal to the SSBP2 LUFS domain. (D) Mutagenesis analysis of LDB1/SSBP2 interface, as shown by MBP pull-down assays. (E) Binding affinities of 3 MBP-tagged LDB1 mutants with SSBP2(1–94) were measured by SPR assays. MBP is a negative control, and the values are presented ± SD.

Fig. 5.

β-Globin transcriptional activity and chromatin looping analysis of LDB1 mutants. (A) Western blots with LDB1 and GFP antibodies. MEL cell differentiation was induced with 1.5% vol/vol dimethyl sulfoxide (DMSO) for 4 d. UMEL, uninduced MEL cells; IMEL, induced MEL cells. (B) Induction of LDB1 KO MEL, MEL, and cells rescued by expression of LDB1 mutants from lentiviruses. Red-colored pellets are indicative of β-globin (hemoglobin) production in induced cells. (C) β-Globin expression after induction. LDB1 KO MEL cells are rescued with wild-type LDB1, mut1, mut2, or mut4. UMEL, uninduced MEL; IMEL, induced MEL. (D) Long-range interactions in the β-globin locus. A chromosome conformation capture (3C) analysis was performed for cells expressing LDB1 and LDB1 mutants. Relative interaction frequency between the HS2 as anchor fragment and β-globin. Bh1 locus served as a control. Error bars in the figures represent standard error (SEM; ***P < 0.001, ****P < 0.0001).

Fig. 6.

The LDB1 NTF2-like subdomain contains a potential small-molecule binding pocket. (A and B) Cartoon and surface representation of LDB1 NTF2-like subdomain in stereo view. (C) Structural superposition of different NTF2-like folds: those of LDB1 (in orange); LinA (in yellow; PDB code 3A76); CDL2.2, a computationally designed Vitamin-D3 binder (in gray; PDB code 5IEN); and NTF2 (in cyan; PDB code 1U5O). The ligand from 5IEN is colored in red. (D) This pocket is covered by the LDB1 ₂₂₉NITRCGL₂₃₅ motif between the H5 in LDB1 DD and the LCCD H6. A, B, and D have the same orientation. (E) A cutaway illustration of the potential ligand-binding pocket of the LDB1 NTF2-like subdomain. Sidechains of I230, T231, and L235 cover this pocket in the LDB1/SSBP2 complex structure.