Montoya Group – University of Copenhagen

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CPR > Research > Protein Structure & Function Program > Montoya Group

Montoya Group - Macromolecular Crystallography

 

The Macromolecular Crystallography group studies the structure of macromolecules involved in cell cycle and genome stability and their interactions. Using this approach, we can explore basic mechanistic questions regarding protein function and the evolutionary relationships between cellular components, and we may discover new and better targets for developing novel therapeutics against diseases such as cancer. However, the current lack of knowledge of macromolecules at the atomic level hampers our full understanding of these biological processes, thereby hindering translational advances.

We apply our well-established expertise to combine biophysical and biochemical assays with structural approaches such as X-ray crystallography and electron microscopy to pursue our research goals.

First row from the left: Guillermo Montoya, Dan Yang Wang, Pablo Mesa, Stefano Stella, Dario Aponte, Nicholas Egholm Sofos, Nika Jachowicz, Nehar Mortuza, Elisabeth Bragado Nilsson, Bhargav Saligram, Diana Zina Kowalick, Pablo Alcón

Challenges and research aims

Macromolecules underlie all biological processes and play either dynamic roles in catalysis or signaling or static roles in scaffolding or information storage. The focus of our group is the molecular understanding of the role played by macromolecules involved in cell cycle and genome integrity related processes. 

Macromolecular machines involved in cell division dynamics and control

The assemblies of these macromolecules, the molecular machines of the cell, perform the majority of cellular functions. Obviously the current information of cellular structures contains limited knowledge both of the cellular organization, localization and the action mechanisms of these molecular machines. There is a lack of information 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 into the design principles of living systems at levels that span from basic mechanistic questions regarding protein function to the evolutionary relationships between cellular components. Recent methodological improvements allowed the structure solution at atomic or near to atomic resolution of complex molecular machines.

Video 1.- Crystal structure of the CCT/TriC-tubulin complex

Overall structure of the CCT-Tubulin complex.The structure is rotated perpendicular to the 8-fold axis to show the experimental electron density of the tubulin molecule inside the chaperonin cavity. (Muñoz et al., Nature Structural & Molecular Biology 2011)

Besides, high-resolution structures solved by X-ray or NMR, of individual subunits and/or their domains, can be docked into medium-low resolution electron microscopy maps, unravelling key information about the structure and function of multi-subunit complexes and the protein-protein interactions of these molecular machines. During cell division different proteins and protein complexes form assemblies in a coordinated manner in order to perform essential functions.

Structural Biology of Genome Editing Tools
The engineering of protein–DNA interactions and the capability of cutting the genome precisely open the possibility to provide “toolkits” to modify the genome. The combination of precise DNA-binding domains with different activities that may act on DNA provides a potentially useful means to modify the genome. The members of Class 2 CRISPR-Cas are composed by a single protein-RNA complex which has been harnessed for gene targeting. The ability to redesign the guide RNA sequence of these ribonucleoproteins, to target specific DNA sites, has resulted in a powerful method for genome modification in multiple biomedical and biotechnological applications.

Video 2. Detailed view of Cpf1 PAM recognition and DNA unzipping

The video illustrates the structure of Cpf1, showing how the LKL helix in the PAM-interacting domain is inserted in between the two DNA strands after the PAM site, initiating the double helix melting and acting as a molecular ruler to determine the right size of the cleaved product. For more info see Stella, Alcon et al., Nature 2017

The engineering of homing endonucleases and alternative scaffolds, such as BuDs or TALE, has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Customized templates targeting selected human genes can excise or correct regions of genes implicated in diseases, thereby representing important tools for intervention in eukaryotic genomes.

Video 3.- Detailed mechanism of double strand break generation by an endonuclease

This video displays the molecular mechanism of a DNA double strand break generated by I-DmoI. The reaction was observed step by step by using X-ray crystallography (Molina, Stella et al., Nature Structural & Molecular Biology, 2015). Understanding how these DNA editing tools work could be used to redesign them, thus generating more specific and precise instruments recognizing and cleaving the genome in selected regions. These tools could be used in possible biomedical applications such as the one shown in the video. See a longer version in  youtube https://www.youtube.com/watch?v=YFutF3Cqk3U

Recent selected publications

 

Stella S, Alcón P, Montoya G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature. (2017) May 31. doi: 10.1038/nature22398.

Molina R, Stella S, Redondo P, Gomez H, Marcaida MJ, Orozco M, Prieto J, Montoya G. Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nature Structural & Molecular Biology (2015) Jan;22(1):65-72. doi: 10.1038/nsmb.2932.

Mortuza G., Cavazza T., Garcia-Mayoral MF, Hermida D., Peset I., Pedrero J.G., Lyngsø, J. Marta Bruix, Pedersen, J.S, Vernos, I. and Montoya, G. The XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation. Nature Communications (2014) DOI: 10.1038/ncomms6072.

Muñoz, I.G., Yébenes, H., Zhou M., Mesa, P. Serna, M. Bragado-Nilsson, Beloso, A., E. de Carcer G., Malumbres M., Robinson C.V., Valpuesta, J.M. & Montoya G. Crystal structure of the mammalian cytosolic chaperonin CCT in complex with tubulin. Nature Structural & Molecular Biology (2011) Jan; 18(1):14-9.

Yebenes H, Mesa P, Muñoz I, Montoya G*, Valpuesta JM*. Chaperonins: Two rings for folding. Trends in Biochemical Sciences (2011), 36, 424-432.

P. Redondo, J. Prieto, I. Muñoz, A. Alibés, F. Strichter, L. Serrano, S., P. Duchateau, F. Paques, F. Blanco & G. Montoya. Molecular basis of recognition and cleavage of the human Xeroderma pigmentosum group C gene by engineered homing endonuclease heterodimers. Nature (2008). Nov 6;456(7218):107-1