SPANISH NATIONAL CANCER RESEARCH CENTRE, CNIO 79ANNUAL REPORT 2015 78

Structural Biology and Biocomputing programme | MACROMOLECULAR CRySTALLOgRAPHy gROUPVice-direction of BaSic reSearch

MACROMOLECULAR CRYSTALLOGRAPHY GROUP

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Macromolecules and their interactions underlie all biological processes and play either, dynamic roles in catalysis or signalling, or static roles in scaffolding or information storage. Our Group focuses on the molecular understanding of the role played by macromolecules involved in oncogenic processes. There is an information gap between our current knowledge and our understanding of the molecular mechanisms that govern the function of different cellular machines. Structural determination reveals an unparalleled view of the design principles of living systems at levels that span from basic mechanistic questions regarding protein function, to the evolutionary relationships between cellular components. To achieve this, our work focuses on the structural and dynamic interactions of these biomolecules and their complexes.

“ We have visualised, for the first time, the dynamics of DNA phosphodiester hydrolysis by an endonuclease.”

Guillermo MontoyaGroup Leader

Staff ScientistJesús Prieto

Post-Doctoral FellowRafael A. Molina ( until September )

TechnicianIgor Yefimenko ( until September )

SPANISH NATIONAL CANCER RESEARCH CENTRE, CNIO 81ANNUAL REPORT 2015 80

Structural Biology and Biocomputing programme | MACROMOLECULAR CRySTALLOgRAPHy gROUPVice-direction of BaSic reSearch

Mitotic complexes

Cellular growth and division are regulated by an integrated protein network that ensures the genomic integrity of all eukaryotic cells during mitosis. Microtubules play an important role in several cellular processes, particularly in the formation of the mitotic spindle. The regulation of microtubule dynamics during mitosis is key for spindle formation. Spindle defects, arising from failures in setting up the microtubules, lead to chromosomal instability and aneuploidy, a common cause of tumour development. One of the most effective strategies for cancer treatment so far has been to interfere with the highly dynamic mitotic spindle microtubules ; tubulin remains the

most successful spindle targeted molecule in cancer. To date, novel anti-mitotic agents have demonstrated limited efficacy in clinical trials and classical anti microtubule drugs are still considered as being the best approach for cancer therapy. We are attempting to dissect the molecular working mechanism of CCT/TRiC, the molecule responsible for the folding of tubulin and actin, which are the essential building blocks of the cytoskeleton. This molecular machine is essential for sister chromatid separation through the folding of key anaphase promoting factor subunits, such as Cdc20. Using a hybrid approach, we are aiming to dissect the molecular recognition of these key substrates by the chaperonin. s

reSearch highlightS

Structural design of protein-DNA interactions for gene targeting

We have observed, for the first time, the hydrolytic reaction performed by a specific endonuclease on its target DNA ; we watched how an endonuclease generates a double-strand break in a DNA molecule following a two-metal ion mechanism. To investigate this process we developed a procedure to slow down the enzymatic reaction. This method allowed us to monitor the kinetics of enzyme catalysis using a time-resolved crystallography approach capturing the structures of successive reaction intermediates. Thus, we have provided a uniquely detailed view of the dynamic processes of this key biological reaction. Our work interlaced structural and molecular dynamic analyses to dissect the hydrolysis of a phosphodiester bond, thereby precisely defining the catalytic mechanism of an endonuclease. We have solved more than 150 crystal structures to obtain key snapshots of different catalytic stages, showing the orchestrated conformational changes in the amino acids, nucleotides and metals during catalysis. This work provides the first ‘ live ’ and visual proof of this key biological mechanism ( FIGURE 1 ). This information may be used to engineer even more precise ‘ cutters ’. These scaffolds can present new perspectives for a wide range of applications, such as the correction of mutations linked to monogenic inherited diseases. Our Group has solved

the crystallographic structures of different variants, revealing the molecular basis of new target DNA recognition domains. In addition, we have shown that the repair of the damaged gene can occur at its locus in human cells, thereby opening up avenues for the identification of possible therapeutic applications.

The telomeric nucleosome

The telomere is a specialised region of the chromatids that contains repetitive DNA sequences. This ‘ buffer ’ DNA is truncated during chromosome replication and needs to be further expanded by the telomerase. Different proteins and protein-DNA complexes are implicated in the assembly of a supramolecular structure of the telomeric chromatin that caps the telomere end avoiding the activation of the DNA Damage Response and harmful chromosome fusion. One of the essential protein components involved in this assembly is the shelterin complex, consisting of 6 different proteins : TRF1, TRF2, TIN2, POT1, TPP1 and RAP1. The current literature contains scarce knowledge of these mechanisms. In this proposal we aim to decipher the molecular basis of shelterin telomere capping and telomere organisation ( FIGURE 2 ). These data will help us to understand how the loss of telomere protection contributes to genome instability.

Figure 1 ( A ) Detailed views of the active centre, with Fo– Fc( 0 d–10 d ) omit maps superimposed onto their corresponding refined structures. The omit maps ’ density is contoured at 5σ. The reaction time course displays the 7 different structural reaction intermediates captured in this work. ( B ) Sketch of the reaction depicting the entrance of cations and the cleavage of the bonds of the noncoding and coding strands

during the course of the reaction. ( C ) Mn 2+ anomalous maps of the 7 reaction intermediates, displaying the sequential entrance of the cations in sites A, B and C, and the exit of the crucial metal ion in the central site after DSB generation. All anomalous maps show density contoured at 6σ except in state 3, for which density is contoured at 4σ to show the entrance of Mn 2+ in site C.

Figure 2 Electron density map at 1s of the crystal structure of the telomeric

nucleosome particle at 3.15 Å resolution in complex with associated factors.

∞ PUBLICATIONS

∞ Molina R, Stella S, Redondo P, Gomez H, Marcaida MJ, Orozco M, Prieto J, Mon-toya G ( 2015 ). Visualizing phosphodi-ester-bond hydrolysis by an endonuclease. Nat Struct Mol Biol 22, 65-72.

∞ De Biasio A, de Opakua AI, Mortuza GB, Molina R, Cordeiro TN, Castillo F, Villate M, Merino N, Delgado S, Gil-Cartón D, Luque I, Diercks T, Bernadó P, Montoya G, Blanco FJ ( 2015 ). Structure of p15( PAF )-PCNA

complex and implications for clamp sliding during DNA replication and repair. Nat Commun 6,6439.

∞ Comino-Méndez I, Leandro-García LJ, Montoya G, Inglada-Pérez L, de Cubas AA, Currás-Freixes M, Tysoe C, Izatt L, Letón R, Gómez-Graña Á, Mancikova V, Apellániz-Ruiz M, Mannelli M, Schiavi F, Favier J, Gimenez-Roqueplo AP, Timmers HJ, Roncador G, Garcia JF, Rodríguez-An-tona C, Robledo M, Cascón A. ( 2015 ). Functional and in silico assessment of

MAX variants of unknown significance. J Mol Med 93, 1247-1255.

∞ Molina R, Marcaida MJ, Redondo P, Ma-renchino M, Duchateau P, D’Abramo M, Montoya G, Prieto J ( 2015 ). Engineering a nickase on the homing endonuclease I-DmoI scaffold. J Biol Chem 290, 18534-18544.

∞ Molina R, Redondo P, López-Méndez B, Villate M, Merino N, Blanco FJ, Valton J, Grizot S, Duchateau P, Prieto J, Montoya G ( 2015 ). Crystal Structure of the Hom-

ing Endonuclease I-CvuI Provides a New Template for Genome Modification. J Biol Chem 290, 28727-28736.

∞ AWARDS AND RECOGNITION

∞ Co-organiser, EMBO Workshop “ Cell di-vision : molecular machineries and cancer targeted therapies ” with co-sponsorship by UNIA.