Establishing the LMO4-binding region of DEAF1.

<p><b>A.</b> Schematic domain structure of the mouse DEAF1 protein containing a DNA-binding SAND (Sp100, AIRE-1, NucP41/75, DEAF11), a Helix-Loop-Helix (HLH) domain, a predicted coiled coil region (depicted as a helix), a protein-binding MYND (myeloid translocation protein 8, Nervy, DEAF1) domain, a nuclear localization signal (NLS) and a nuclear export signal (NES). The previously identified LMO4-binding region (335–545) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039218#pone.0039218-Sugihara1" target="_blank">[4]</a> is indicated (thin grey line). <b>B.</b> Yeast two-hybrid experiments where <i>Saccharomyces cerevisiae</i> (AH109) were co-transformed with full-length DEAF1 fused to a transcriptional activator domain (pGAD10) and LMO4 fused to a DNA binding domain (pGBT9). Co-transformants were serially diluted and spotted on growth (−L/−W; growth) and high stringency interaction plates (−L/−W/−H/−A; selection). Left-most panels show controls. Schematic on right shows corresponding domain truncations of DEAF1 constructs used in the assays. Growth of yeast or its absence on selection plates indicates an interaction (ticks) or an abrogation of the interaction (crosses) with LMO4 respectively. <b>C.</b> DEAF1 internal deletion mutants were tested for interaction with LMO4 by yeast two-hybrid assays; interactions are represented as above. <b>D.</b> Yeast two-hybrid data for DEAF1/LMO4 interactions to assay replacement of the DEAF1 coiled coil domain by the dimeric GCN4 leucine zipper. Selection was medium/high stringency (−L/−W/−H+3AT)/(−L/−W/−H/−A). Yeast two-hybrid spot test results are shown. Three dilutions (A_600nm = 0.2, diluted serially 2×1-in-10) are spotted left to right to show differences in growth under each selection condition.</p

Characterising DEAF1 and the coiled coil domain.

<p><b>A.</b> Schematic showing DEAF1 constructs used in yeast two-hybrid self-association experiments. Selection was medium/high stringency as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039218#pone-0039218-g001" target="_blank">Figure 1D</a>; +++ indicates strong growth, - indicates no growth, ND indicates not determined. <b>B.</b> SEC-MALLS analysis of full length and DEAF1_335–485 constructs (left panel) and DEAF1_404–479 and the coiled coil domain (right panel). DEAF1 proteins (∼200 µg) were applied to a Superose 12 column with an in line MALLS detector to determine weight-averaged molecular weight in solution. The elution (continuous line) and light-scattering (▪) are shown. <b>C.</b> Summary of the theoretical monomeric and experimentally determined molecular weight of DEAF1 proteins in A and B were used to calculate the oligomeric state. <b>D.</b> Far-UV circular dichroism spectropolarimetry (CD) spectrum of the DEAF1 coiled coil domain.</p

Nuclear localisation of EGFP-NLS4-DEAF1_404–479 in the presence of LMO4.

<p><b>A.</b> DEAF1 construct in pEGFP-C2 that was used for transfection. It has an N-terminal EGFP tag followed by the altered DEAF1 NLS4 and DEAF1_404–479. The NLS4 and NES protein sequences and DEAF1 numbering are shown. <b>B.</b> HEK293 cells grown on cover slips in 6 well plates were transfected with a total of 4 µg of DNA: control pEGFP (panel 1), 2 µg EGFP-DEAF1_404–479+2 µg empty pCMV vector (panel 2), 2 µg EGFP-NLS4-DEAF1_404–479+2 µg empty pCMV (panel 3) and EGFP-NLS4-DEAF1_404–479+2 µg pCMV LMO4 (panel 4). After 24 h transfection, cells were fixed with paraformaldehyde and nuclei stained with Hoechst dye. Cells were imaged for EGFP fluorescence (green) and nuclear staining (blue) by fluorescence microscopy. <b>C.</b> Quantification of A. The two-dimensional areas of <i>n</i> = 8 fields of view were measured for % nuclear localisation of EGFP-NLS4-DEAF1_404–479 in the presence and absence of LMO4. Difference is statistically significant as <i>p</i><0.05.</p

The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4

<div><p>LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4_LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4_LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.</p></div

Yeast two-hybrid and mutagenic analysis of LMO4-DEAF1 binding.

<p>(A) Data showing the interaction of DEAF1_45–566 (DEAF1) or control (empty) with the tandem LIM domains of LMO4 (LMO4), the isolated LIM domains of LMO4 (LIM1 and LIM2) or a pre-formed LMO4•LDB1_LID complex. These were spotted onto low-stringency interaction plates or growth control plates. “Empty” refers to pGAD10 vector lacking an insert. (B) Summary of yeast two-hybrid work. Surface residues of LMO4 that when mutated strongly affected (red), attenuated (orange) or had no effect (blue) on interaction with DEAF1 are mapped onto the structure of LMO4•LDB1_LID (1RUT). Non-mutated residues are in white, and LDB1_LID is shown as dark sticks. (C) Mutagenic scanning of the minimal LMO4-binding domain of DEAF1 (in the DEAF1_{404–438_457–479} construct). Residues in DEAF1_404–438 were systematically mutated to alanine or glycine in sets of three (or two) as indicated and analysed for binding to LMO4 using yeast two-hybrid assays. Co-transformants were spotted onto selective media (low, medium and high stringency plates) as well as growth control plates. The sequence of DEAF1 is coloured according to whether the mutation strongly affected binding (red), attenuated binding (orange) or had no effect (blue) compared to wild-type positive control on each plate (“wt”). Thick white lines indicate separate plates.</p

Engineering tethered LMO4_LIM2•DEAF1_404–418 and DEAF1_404–418•LMO4_LIM2 complexes.

<p>(A) Schematics of full-length LMO4 (blue) and DEAF1 (orange) and engineered ‘intramolecular complexes’ of LMO4_LIM2 and DEAF1_404–418. The complexes are tethered via a glycine-serine linker (red) either from the C-terminus of LMO4 to the N-terminus of DEAF1 or vice versa. SAND, coiled-coil (CC) and MYND domains, and nuclear localisation (NLS) and nuclear export (NES) signals in DEAF1 and the LIM1 and LIM2 domains in LMO4 are indicated. (B) MALLS analysis of tethered constructs as indicated; protein concentrations at the detectors are 30 µM. Lines represent the refractive index and calculated molecular weights are shown as symbols. Monomeric BSA (blue) was used as a standard. (C) ¹⁵N-HSQC spectra of LMO4_LIM2•DEAF1_404–418 (black) and DEAF1_404–418•LMO4_LIM2 (red) were recorded in 20 mM sodium acetate at pH 5.0, 35 mM NaCl and 0.5 mM TCEP-HCl at 298 K on a 600 MHz spectrometer.</p

The LMO4-binding domain from DEAF1 is disordered in solution.

<p>(A) The sequence of L4-DEAF1 includes residues 404–438 of DEAF1 (bold), a T435D point mutation (underlined) and a polyproline C-terminal tail (PPPPPR). The two N-terminal residues (GS) are an artefact of the plasmid and remain after treatment with thrombin. (B) ¹⁵N-HSQC spectrum of L4-DEAF1 (160 µM) was recorded in 20 mM sodium acetate at pH 5.0 and 35 mM NaCl at 298 K on a 600 MHz spectrometer equipped with a TCI-cryogenic probehead. (C) The far-UV CD spectrum of L4-DEAF1 (40 µM) dissolved in 20 mM Tris-acetate at pH 8.0 and 50 mM NaF.</p

Relaxation analysis of LMO4_LIM2•DEAF1_404–418.

<p>(A) Longitudinal (<i>T</i>₁), (B) transverse (<i>T</i>₂) relaxation time constants, (C) heteronuclear NOEs, calculated as the ratio of peak intensities with and without proton saturation, all at 600 MHz. (D) Lipari-Szabo (S²) parameters for each assigned backbone amide group in LMO4_LIM2•DEAF1_404–418 calculated from data recorded at 600 MHz and 800 MHz, using the program relax. Error bars represent one standard deviation from the curve fit for each residue. Background colours indicate regions belonging to LMO4 (blue), DEAF1 (yellow) or the glycine-serine linker (G/S; grey).</p

LMO4 is a protein-protein interaction network hub linking multiple cellular processes.

<p>Protein-protein interaction network assembled from data reported for mouse and human LMO4 proteins from the STRING protein-protein interaction database, plus additional papers cited in the introduction. Bold lines indicate protein-protein interactions that have been characterised structurally. Other lines indicate reported interactions that have different levels of evidence and some of these lines may represent indirect interactions. Proteins are loosely grouped into cellular processes.</p

NMR restraints and refinement statistics for LMO4_LIM2DEAF1_404–418.

a<p>There were no dihedral angle violations >5°.</p>b<p>Full parameter and topology files are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109108#pone.0109108.s001" target="_blank">File S1</a>.</p>c<p>Regions of LMO4 between residues 86–139 and of DEAF1 between residues 404–414 including S208 of the glycine-serine linker were considered to be structured because the residues contained within had sum of angle order parameters (φ + ψ)>1.8 except for residues 103–105 of LMO4 and residues 404, 406 and 407 of DEAF1.</p>d<p>Distance violations were restricted to disordered regions of the protein.</p><p>NMR restraints and refinement statistics for LMO4_LIM2DEAF1_404–418.</p