Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins

Analysis of the Nse3/MAGE-Binding Domain of the Nse4/EID Family Proteins

<div><h3>Background</h3><p>The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket.</p> <h3>Methodology/Principal Findings</h3><p>Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family.</p> <h3>Conclusions/Significance</h3><p>We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.</p> </div

MAGE proteins bind to Nse3-binding domain of the NSE4b protein.

<p>The GST-His-S-tagged fragment of human NSE4b(106 - 135) was bound to S-protein agarose beads (lanes 1–3) and then incubated with <i>in vitro</i> translated class I (panel <b>A</b> and <b>B</b>, lanes 1–6) and/or class II (panel <b>C</b> and <b>D</b>, lanes 1–6) MAGE proteins: MAGEA1 (aa 1-309; panel <b>A</b>), MAGEC2 (aa 6-373; panel <b>B</b>), MAGED4b (aa 1-741; panel <b>C</b>) and necdin (aa 1-321; panel <b>D</b>). The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the GST-His-S-tagged protein was analyzed by immunoblotting with anti-His antibody and the <i>in vitro</i> translated proteins were measured by autoradiography. Control, GST-His-S-tag protein (lanes 4–6).</p

NSE4/EID proteins interact with MAGE proteins through the Nse3/MAGE-binding domain.

<p>(<b>A.</b>) Alignments of the five members of the human NSE4/EID family: EID1, EID2, EID2b, EID3/NSE4b and NSE4a. The red box indicates Nse3/MAGE-binding domain; hatched and crosshatched boxes indicate kleisin and kleisin-like motifs, respectively (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone-0035813-g001" target="_blank">Fig. 1B and C</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone.0035813-Palecek1" target="_blank">[10]</a>); grey boxes indicate regions of homology between EID1 and 2, respectively. (<b>B. and C.</b>) GST-His-S-tagged fragments of each of the NSE4/EID family members were pre-bound to S-protein agarose beads and incubated with <i>in vitro</i> translated MAGEA1 (aa 1-309; panel <b>B</b>) and necdin (aa 1-321; panel <b>C</b>). The indicated NSE4/EID proteins correspond to the MAGE-interacting domain of EID1(aa146-177), EID2(aa197-225), EID2b(aa135-161), NSE4b/EID3(aa106-135) and NSE4a(aa150-179). In lanes 8-12, immunoblotting of bound fragments are shown in the upper panel and bound radioactive MAGE proteins (MAGEA1 and necdin) in the lower panel. Control, GST-His-S protein alone.</p

Nse3/MAGE-binding domain within the Nse4/EID family.

<p>(<b>A.</b>) The His-MBP-tagged fragment of yeast Nse3 (aa 200 - 307) was pre-bound to amylose-beads (lanes 1–3, 7–9 and 10–12) and then incubated with <i>in vitro</i> translated fragments spanning aa 1 to 110 (lanes 1–3), aa 1 to 77 (lanes 7–9) and/or aa 75 to 104 (lanes 10–12) of the yeast Nse4 protein. The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the His-MBP-tagged protein was analyzed by immunoblotting with anti-His antibody and the <i>in vitro</i> translated proteins were measured by autoradiography. I, input (5% of total); U, unbound (5%); B, bound (40%). Control, no His-MBP-tagged protein present. Alignment of Nse4 (<b>B.</b>) and EID (<b>C.</b>) subfamilies. The orthologs are from <i>Schizosaccharomyces pombe</i> (<i>S.p.</i>), <i>Aspergillus nidulans</i> (<i>A.n.</i>), <i>Neosartorya fischeri</i> (<i>N.f.</i>), <i>Aspergillus terreus</i> (<i>A.t.</i>), <i>Aspergillus clavatus</i> (<i>A.c.</i>), <i>S. cerevisiae</i> (<i>S.c.</i>), <i>Danio rerio</i> (<i>D.r.</i>), <i>Xenopus leavis</i> (<i>X.l.</i>), <i>Galus galus</i> (<i>G.g.</i>), <i>Monodelphis domestica</i> (<i>M.d.</i>), <i>Dasypus novemcinctus</i> (<i>D.n.</i>), <i>Canis lupus familiaris</i> (<i>C.f.</i>), <i>Mus musculus</i> (<i>M.m.</i>), <i>Homo sapiens</i> (<i>H.s.</i>). The EID homologs are present only in some mammals. Red box, Nse3/MAGE-binding domain; blue plus, mutation not affecting interaction; red minus, mutation disrupting the interaction. Amino acid shading represents groups conserved across the family: <i>dark green</i>, hydrophobic and aromatic; <i>light green</i>, polar; <i>pink</i>, acidic; <i>blue</i>, basic; all glycine and proline residues are highlighted in <i>yellow</i>.</p

Docking model of the EID2-MAGEC2 heterodimer.

<p>Homology modelling was used to generate the predicted MAGEC2 (aa133-336) and EID2 (¹⁹⁷QRNPHRVDLDILTFTIALTASEVINPLIEE²²⁶) structure. (<b>A.</b>) Ribbon representation of the predicted MAGEC2 3D structure model (blue; helices indicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone.0035813-Hudson1" target="_blank">[9]</a>). (<b>B.</b>) MAGEC2 surface view (blue) with docked EID2 peptide (yellow; ribbon representation). (<b>C.</b>) Stereoscopic detailed view of the MAGEC2 pocket with bound EID2 peptide. The EID2 residues involved in the binding to MAGEC2 are indicated in red (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone-0035813-g005" target="_blank">Fig. 5A</a>). The central EID2 amino acid residues (L205, D206, I207, L208, F210, I212 and L214) are in physical contact with the MAGEC2 pocket surface (formed by H4, H5 and H8). The N-terminus of the Nse3/MAGE-binding domain makes contact through the essential residues R198 and R202 (only R198 is labeled) to the MAGEC2 loop region (between H5 and H6). The D204 residue protruding to the solvent is black labeled.</p

Analysis of EID2 binding to MAGE proteins.

<p>(<b>A.</b>) Quantification of relative binding of the MAGEC2(129-339) protein (red columns) to the EID2 protein-based synthetic mutant peptides (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone-0035813-t001" target="_blank">Table 1</a>) using the PEPSCAN-ELISA method. Results show mean ± SEM of 3 independent measurements. His-hTRF2 protein (white column) was used in the control experiment. (<b>B. to D.</b>) The short (biotin-SGSG-²⁰¹HRVDLDILTFTIALTAS²¹⁷) and long (biotin-SGSG-¹⁹⁷QRNPHRVDLDILTFTIALTAS²¹⁷) EID2 peptides were pre-bound to the streptavidin-agarose beads and then incubated with <i>in vitro</i> translated MAGEC2 (aa 6-373; C2 in panel <b>B.</b>), MAGEA1 (aa 1-309; A1 in panel <b>C.</b>) and/or necdin (aa 1-321; nd in panel <b>D.</b>) protein, respectively. (<b>E.</b>) Wild type and selected EID2 mutant peptides (as indicated) were pre-bound to the streptavidin-agarose beads and then incubated with <i>in vitro</i> translated necdin protein. The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the <i>in vitro</i> translated proteins was measured by autoradiography. Control, no peptide.</p