|Structural Biology and Biophysics|
|Protein structure and interactions
Nuclear Magnetic Resonance (NMR)
Our research is focused on the structural analysis of proteins and its complexes in solution. NMR has the potential to determine the structure of the protein in solution, detect ligand binding, map the binding site and quantify the affinity of the interaction. We use this methodology to characterise and modulate protein-protein interactions involved in chromatin regulation and signalling.
ING4, a member of the Inhibitor of Growth family of tumor suppressors, contains a C-terminal Plant Homeodomain (PHD) that binds to histone H3 tail methylated at Lys4 (H3K4me3). ING4 is involved in transcription activation through its interaction with the Histone AcetylTransferase complex HBO1. Binding affects the PHD backbone dynamics, and the specificity for the differentially methylated histone H3 tails is entropy driven. Full length ING4 forms homodimers through its coiled coil N-terminal domain. The central region of ING4 is disordered and flexible, and does not directly interact with p53 or does it with very low affinity, in contrast with previous findings. The two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 with the same affinity as the isolated PHD finger. These results show that ING4 is a bivalent reader of the chromatin H3K4me3 modification and suggest a mechanism for enhanced targeting of HBO1 complex to specific chromatin sites.
Gadd45α (Growth Arrest and DNA Damage-inducible α) is a nuclear protein transcriptionally regulated by p53. The interactions of Gadd45α with other proteins play a central role in DNA repair, cell cycle control and apoptosis. The solution structure of human Gadd45α shows an α/β fold with a five-stranded mixed β-sheet at the core and five helices surrounding it. We have seen by NMR that the previously reported interaction with PCNA (Proliferating Cellular Nuclear Antigen, an essential factor for DNA replication and repair) is transient and very weak, while Gadd45α binds to the N-terminal non-kinase domain of the mitotic kinase Aurora A.
Meganucleases recognize long DNA sequences and produce double strand breaks (DSB) at single sites in whole genomes. These rare cutting endonucleases can be engineered to repair defective genes ex vivo by promoting efficient gene targeting through DSB-induced homologous recombination. We have contributed to the development of novel meganucleases that cleave DNA from the human XPC and RAG1 genes (involved in Xeroderma Pigmentosum and in SCID diseases, respectively) in vitro and in human cells with very low genotoxicity.
|Molecular basis of xeroderma pigmentoxum group C DNA recognition by engineered meganucleases. P Redondo, J Prieto, I G. Muñoz, A Alibés, F Stricher, L Serrano, J-P Cabaniols, F Daboussi, S Arnould5, C Perez, P Duchateau, F Pâques, F J Blanco* & G Montoya* (2008) Nature, 456, 107-111.|
|The Dimeric Structure and the Bivalent Recognition of H3K4me3 by the Tumor Suppressor ING4 Suggests a Mechanism for Enhanced Targeting of HBO1 Complex to Chromatin. Palacios A, Moreno A, Oliveira BL, Rivera T, Prieto J, García P, Fernández-Fernández MR, Bernadó P, Palmero I, Blanco FJ (2010) J Mol Biol 396, 1117-1127.|
|Solution Structure of Human Growth Arrest and DNA Damage 45 (Gadd45) and its Interactions with Proliferating Cell Nuclear Antigen (PCNA) and Aurora A Kinase Sánchez R, Pantoja-Uceda D, Prieto J, Diercks T, Marcaida MJ, Montoya G, Campos-Olivas R, Blanco FJ (2010) J Biol Chem 285, 22196-22201.|
|Reduced stability and increased dynamics in the human proliferating cell nuclear antigen (PCNA) relative to the yeast homolog. A De Biasio, R Sánchez, J Prieto, M Villate, R Campos-Olivas, FJ Blanco (2011) PLoS One 6, e16600.|
|Transient Protein-DNA/RNA interactions. FJ Blanco, G Montoya (2011) FEBS J 278, 1643-1650..|