Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families Jessica J. R. Hudson1., Katerina Bednarova2,3., Lucie Kozakova3, Chunyan Liao1¤, Marc Guerineau2, Rita Colnaghi1, Susanne Vidot1, Jaromir Marek2,3, Sreenivas R. Bathula4, Alan R. Lehmann1*, Jan Palecek2,3* 1 Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom, 2 Functional Genomics and Proteomics, Masaryk University, Brno, Czech Republic, 3 Central European Institute of Technology, Masaryk University, Brno, Czech Republic, 4 National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic Abstract Background: 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. Methodology/Principal Findings: 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. Conclusions/Significance: 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. Citation: Hudson JJR, Bednarova K, Kozakova L, Liao C, Guerineau M, et al. (2011) Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families. PLoS ONE 6(2): e17270. doi:10.1371/journal.pone.0017270 Editor: Michael Lichten, National Cancer Institute, United States of America Received November 29, 2010; Accepted January 25, 2011; Published February 25, 2011 Copyright: 2011 Hudson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to J.P. from the Grant Agency of the Czech Academy of Sciences (IAA501630902, http://www.gaav.cz/en/) and Institutional support was provided by Czech Ministry of Education, Youth and Sports (MSM0021622415, http://www.msmt.cz/eu-presidency-2009/events). M.G. was supported from Grant Agency of the Czech Republic (204/08/H054, http://www.gacr.cz/international.htm). Work from the ARL lab was funded by an MRC Programme Grant. The authors are grateful to the METACentrum (super)computing facilities at Masaryk University for access under the research intent MSM6383917201. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jpalecek@sci.muni.cz (JP); a.r.lehmann@sussex.ac.uk (ARL) . These authors contributed equally to this work. ¤ Current address: Nuffield Department of Obstetrics and Gynaecology, The Women’s Centre, John Radcliffe Hospital, Oxford, United Kingdom Introduction The SMC5-6 protein complex is one of the three SMC (Structural maintenance of chromosomes) protein complexes present in all eukaryotes. The core of each complex is a SMC protein heterodimer, which is associated with other non-SMC proteins. In the yeasts SMC5-6 is essential for proliferation as well as being involved in the response to different types of DNA damage [1]. It is required to resolve recombination structures [2–4] as well as having an early role in the recombination process in response to replication stalling [5]. In human cells it is required for loading cohesin at sites of double-strand breaks [6] and for telomere maintenance via the alternative lengthening of telomeres (ALT) pathway [7]. In the yeasts SMC5-6 is comprised of 8 components [8–10]. We and others have shown that there are three sub-complexes. In the Smc6-Smc5-Nse2 sub-complex, Nse2/Mms21 is a SUMO ligase and associates with Smc5 [9,11]. The crystal structure of the heterodimer of Nse2 and the interacting fragment of Smc5 has been reported recently [12]. The Nse1-Nse3-Nse4 sub-complex bridges the head domains of the Smc5-Smc6 heterodimer [8,10,13]. Nse4 is the kleisin component of the complex, but Nse3 also binds both Smc5 and Smc6 globular head domains [13]. Nse1 is a RING finger protein with ubiquitin ligase activity [14]. The third sub-complex is made up of Nse5 and Nse6 [10,13], which are less well conserved than the others and there is no obvious sequence identity between them in Saccharomyces cerevisiae [15] and Schizosaccharomyces pombe [10]. With the exception of Nse5 and Nse6, conserved human orthologs of all the SMC5-6 components have been identified and characterised. Nse3 is related to the MAGE (Melanoma-associated PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17270