Research

Protein-protein cross-linking applications

We have applied chemical cross-linking to numerous protein complexes, mostly in collaborative projects with biologists from around the world. Examples include large, multi-subunit complexes such as ribosomes, the chaperonin TRiC/CCT, the target of rapamycin complex 2 or the membrane-embedded ryanodine receptor. Cross-linking data complements high-resolution structural biology techniques such as X-ray crystallography and cryo-electron microscopy and can help to provide a better understanding of structure and function of these large assemblies. Such insights can also be obtained for smaller complexes, such as the cytomegalovirus viral entry complexes and even single, multi-domain proteins such as huntingtin and the chromatin remodeler Alc1, as we have shown.

Selected publications:

• Leitner et al. The molecular architecture of the eukaryotic chaperonin TRiC/CCT, Structure 2012. external page Link
• Greber et al. Architecture of the large subunit of the mammalian mitochondrial ribosome, Nature 2014. external page Link
• Gaubitz et al. Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2, Mol. Cell 2015. external page Link
• Ciferri et al. Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies, PLOS Pathog. 2015. external page Link
• Efremov et al. Architecture and conformational switch mechanism of the ryanodine receptor, Nature 2015. external page Link
• Greber et al. Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation, Cell 2016. external page Link
• Vijayvargia et al. Huntingtin’s spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function, eLife 2016. external page Link
• Lehmann et al. Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC1, Mol. Cell 2017. external page Link
• Martinez-Martin et al., An Unbiased Screen for Human Cytomegalovirus Identifies Neuropilin-2 as a Central Viral Receptor, Cell 2018. external page Link
• Gestaut et al. The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis, Cell 2019. external page Link
• Klatt et al. A precisely positioned MED12 activation helix stimulates CDK8 kinase activity, Proc. Natl. Acad. Sci. USA 2020. external page Link
• Sabath et al. INTS10–INTS13–INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module, Nat. Commun. 2020. external page Link
  
• Arora Verasztó et al. Architecture and functional dynamics of the pentafunctional AROM complex. Nat. Chem. Biol. 2020. external page Link
  

Protein-RNA cross-linking

More recently, we have extended our cross-linking scope to study protein-RNA complexes. This opens up new possibilities to study interactions between these two important classes of biomolecules. In collaboration with Frédéric Allain’s group at ETH we have established CLIR-MS, a method based on photochemical cross-linking that allows the localization of interaction sites at single residue level for amino acids and nucleotides. We are currently working on further improving the method and applying it to target complexes of biological and clinical interest.

Selected publications:

• Dorn et al. Structural modeling of protein-RNA complexes using crosslinking of segmentally isotope labeled RNA and MS/MS, Nat. Methods 2017. external page Link
• Leitner et al. Combining Mass Spectrometry (MS) and Nucleic Magnetic Resonance (NMR) Spectroscopy for Integrative Structural Biology of Protein-RNA Complexes, Cold Spring Harb. Perspect. Biol. 2019. external page Link
  

Data standards

The importance of data and reporting standards in science is often overlooked, but standards increase transparency and fulfil an important role in helping methods gain acceptance in the wider scientific community. Alexander has made contributions to standards established by the HUPO Proteomics Standard Initiative’s MIAPE guidelines and mzIdentML standard. He was also Swiss delegate for the COST Action BM1403 on Native MS and related methods (external page link) that established guidelines and best practices in the fields of native mass spectrometry, hydrogen/deuterium exchange and cross-linking. From this emerged a community-wide study on cross-linking practices, jointly organized by Alexander and Andrea Sinz (Halle).

Selected publications:

• Taylor et al. The minimum information about a proteomics experiment (MIAPE), Nat. Biotechnol. 2007. external page Link
• Vizcaíno et al. The mzIdentML data standard version 1.2, supporting advances in proteome informatics, Mol. Cell. Proteomics 2017. external page Link
• Iacobucci et al. First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study, Anal. Chem. 2019. external page Link
• Berman et al. Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures, Structure 2019. external page Link   
  

Funding sources

We currently receive funding from ETH Zurich (ETH Research Grant), the Strategic Focus Area external page Personalized Health and Related Technologies of the ETH Domain, and the external page Swiss National Science Foundation via the external page NCCR RNA & Disease and the external page National Research Program "COVID-19". Esben Trabjerg was funded by a fellowship from the external page Benzon Foundation.