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

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 LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-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.SJ was funded by an Australian Postgraduate Award (education.gov.au/australian-postgraduate-awards). JPM and JMM were awarded Senior Research Fellowships from the Australian National and Medical Research Council (www.nhmrc.gov.au). This project was funded by an Australian Research Council (www. arc.gov.au) Discovery Project Grant (DP110104332) to JMM and LC

Insight into the Architecture of the NuRD Complex: Structure of the RbAp48-MTA1 Subcomplex

The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex

GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema

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

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

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

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