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

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 pageLinkcall_made
• 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 pageLinkcall_made
  

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 pagelinkcall_made) 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 pageLinkcall_made
• Vizcaíno et al. The mzIdentML data standard version 1.2, supporting advances in proteome informatics, Mol. Cell. Proteomics 2017. external pageLinkcall_made
• Iacobucci et al. First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study, Anal. Chem. 2019. external pageLinkcall_made
• Berman et al. Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures, Structure 2019. external pageLinkcall_made   
  

Funding sources

We currently receive funding from ETH Zurich (ETH Research Grant), the Strategic Focus Area external pagePersonalized Health and Related Technologiescall_made of the ETH Domain, and the external pageSwiss National Science Foundationcall_made via the external pageNCCR RNA & Diseasecall_made and the external pageNational Research Program "COVID-19"call_made. Esben Trabjerg was funded by a fellowship from the external pageBenzon Foundationcall_made.