@article{fee60307de8e4043bf506f18d0b56af6,
title = "Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes",
abstract = "Smad transcription factors are the main downstream effectors of the Transforming growth factor β superfamily (TGFβ) signalling network. The DNA complexes determined here by X-ray crystallography for the Bone Morphogenetic Proteins (BMP) activated Smad5 and Smad8 proteins reveal that all MH1 domains bind [GGC(GC)|(CG)] motifs similarly, although TGFβ-activated Smad2/3 and Smad4 MH1 domains bind as monomers whereas Smad1/5/8 form helix-swapped dimers. Dimers and monomers are also present in solution, as revealed by NMR. To decipher the characteristics that defined these dimers, we designed chimeric MH1 domains and characterized them using X-ray crystallography. We found that swapping the loop1 between TGFβ- and BMP- activated MH1 domains switches the dimer/monomer propensities. When we scanned the distribution of Smad-bound motifs in ChIP-Seq peaks (Chromatin immunoprecipitation followed by high-throughput sequencing) in Smad-responsive genes, we observed specific site clustering and spacing depending on whether the peaks correspond to BMP- or TGFβ-responsive genes. We also identified significant correlations between site distribution and monomer or dimer propensities. We propose that the MH1 monomer or dimer propensity of Smads contributes to the distinct motif selection genome-wide and together with the MH2 domain association, help define the composition of R-Smad/Smad4 trimeric complexes.",
keywords = "5GC-motifs, BMP signaling, Domain-swapping, MH1 domain, Protein-DNA, Smad, Smad chimeras, Smad5, Smad8, Transcription factor",
author = "Lidia Ruiz and Zuzanna Kaczmarska and Tiago Gomes and Eric Aragon and Carles Torner and Regina Freier and Blazej Baginski and Pau Martin-Malpartida and {de Martin Garrido}, Nat{\`a}lia and Marquez, {Jos{\'e} A.} and Cordeiro, {Tiago N.} and Radoslaw Pluta and Macias, {Maria J.}",
note = "Funding Information: We thank Dr. N. Berrow (IRB Barcelona, protein expression unit) for help with some DNA constructs and with protein purification. We also thank the EMBL staff for assistance at the HTX facility (Grenoble), the joint EMBL and ESRF group for access to synchrotron beamlines ID29, ID23-1 and ID23-2 and the staff at The Automated Crystallography Platform staff (IRB Barcelona-CSIC) and at the ALBA synchrotron (Barcelona) for access to the BL13-XALOC beamline. Thanks also go to Dr. J. Massagu? for insightful suggestions and discussions, Drs. M. D?az and M. Vilaseca (Mass Spectrometry Core Facility, IRB Barcelona) for support with the IM-MS data, Dr. M. Navia for suggestions on binding assays, and Dr. B. Brutscher, (Institut de Biologie Structurale, Biomolecular NMR Spectroscopy Group, Grenoble, France) for help with the acquisition of the NMR data at 850 MHz. We also thank J. Cordero for some preliminary experiments. T.G. was a PhD student funded by a Severo Ochoa and by the BBVA. Z.K. R.F, R.P. and B.B were co-funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sk?odowska-Curie COFUND actions of the EMBL, IRB Barcelona and the PROBIST and PREBIST Postdoc and Predoc Programmes (agreements EMBL_291772, IRBPostPro2.0_600404 and PROBIST_754510, and PREBIST_754558). M.J.M is an ICREA Programme Investigator. This work was supported by the Spanish MINECO program (BFU2014-53787-P and BFU2017-82675-P, M.J.M), IRB Barcelona and the BBVA Foundation. Access to the HTX facility at EMBL (Grenoble) was granted by the Horizon 2020 Programme iNEXT of the European Commission (grant 653706, title: Smad complexes), and to the NMR facility (Grenoble) by the Instruct Integrating Biology program (grant 2520, title: Monomer-dimer equilibrium in Smad proteins). Access to Bio-SAXS BM29 was part of the MX-1941 BAG proposal and to ALBA through the BAG proposal 2018092972. We gratefully acknowledge institutional funding from the CERCA Programme of the Catalan Government and from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) through the Centres of Excellence Severo Ochoa award. L.R. and E.A. cloned, expressed and purified all proteins, L.R. R.F. C.T. and N.M. performed EMSA experiments. L.R. T.G. T.N.C. performed and analyzed the SAXS measurements and P.M.M. L.R. and M.J.M. acquired and analyzed the NMR data. L.R. and T.G. analyzed the IM-MS data. P.M.M. analyzed the clustering of DNA motifs in ChIP-Seq data. Z.K. B.B. and R.P. screened crystallization conditions, collected X-ray data, determined the structures and analyzed them with J.A.M. and M.J.M. All authors contributed ideas to the project. M.J.M. and R.P. supervised the project. M.J.M. wrote the manuscript with contributions from all other authors. Funding Information: T.G. was a PhD student funded by a Severo Ochoa and by the BBVA. Z.K., R.F, R.P., and B.B were co-funded by the European Union{\textquoteright}s Horizon 2020 research and innovation programme under the Marie Sk{\l}odowska-Curie COFUND actions of the EMBL, IRB Barcelona and the PROBIST and PREBIST Postdoc and Predoc Programmes (agreements EMBL_291772, IRBPostPro2.0_600404 and PROBIST_754510, and PREBIST_754558). M.J.M is an ICREA Programme Investigator. This work was supported by the Spanish MINECO program (BFU2014-53787-P and BFU2017-82675-P, M.J.M), IRB Barcelona and the BBVA Foundation. Access to the HTX facility at EMBL (Grenoble) was granted by the Horizon 2020 Programme iNEXT of the European Commission (grant 653706, title: Smad complexes), and to the NMR facility (Grenoble) by the Instruct Integrating Biology program (grant 2520, title: Monomer-dimer equilibrium in Smad proteins). Access to Bio-SAXS BM29 was part of the MX-1941 BAG proposal and to ALBA through the BAG proposal 2018092972. We gratefully acknowledge institutional funding from the CERCA Programme of the Catalan Government and from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) through the Centres of Excellence Severo Ochoa award. Publisher Copyright: {\textcopyright} 2021 The Authors",
year = "2021",
month = jan,
doi = "10.1016/j.csbj.2020.12.044",
language = "English",
volume = "19",
pages = "632--646",
journal = "Computational and Structural Biotechnology Journal",
issn = "2001-0370",
publisher = "Research Network of Computational and Structural Biotechnology",
}