Alexander Leitner

Integrative structural biology of protein-RNA complexes using cross-linking mass spectrometry

Giacomo Padroni1, Maria Bikaki2, Mihajlo Novakovic1, Antje C. Wolter1, Simon H. Rüdisser3, Alvar D. Gossert3, Frederic H.-T. Allain1, Alexander Leitner2

1 Department of Biology, Institute of Biochemistry, ETH Zürich, Zurich, Switzerland
2 Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zurich, Switzerland
3 Biomolecular NMR Spectroscopy Platform, ETH Zürich, Zurich, Switzerland

Introduction: Cross-linking approaches that connect reactive sites in protein complexes, organelles and even entire cells constitute some of the central techniques in structural proteomics. The expansion of cross-linking to probe interactions of different classes of biomolecules (for example, of proteins with oligonucleotides, lipids and carbohydrates) may be considered as the next frontier. Using stable isotope labeling of RNA, we have previously shown that protein-RNA binding interfaces can be characterized at up to single amino acid and nucleotide resolution. In this work, we show how such information can be combined with other experimental and computational methods to unravel interactions between the nucleocapsid protein of SARS-CoV-2 and part of its viral genome, the s2m element.

Methods: Protein-RNA cross-linking was induced by irradiation with 254 nm UV light. Cross-linked complexes were processed using standard workflows, including digestion with RNases A and T1 and trypsin as the protease. Peptide-RNA conjugates were enriched by titanium dioxide affinity chromatography and enriched samples were analyzed by data-dependent LC-MS/MS on a Thermo Orbitrap Fusion Lumos instrument. Data was analyzed by xQuest/RNxQuest. NMR spectroscopy was performed on Bruker instruments of different field strengths equipped with cryoprobes. Docking was performed using HADDOCK 2.4.

Preliminary data: The s2m element has previously been shown to interact with the nucleocapsid (N) protein of SARS-CoV-1 and -2. It is presumed that this interaction plays important roles in viral packaging and replication. To obtain a model of the N-s2m interaction, we followed an approach that combines NMR spectroscopy and CLIR-MS (cross-linking of stable isotope labeled RNA coupled to mass spectrometry). NMR and CLIR-MS data were generated from complexes of the two RNA-binding domains of the N protein, the N-terminal and the C-terminal domain (NTD and CTD, respectively). Restraints obtained from NMR (through chemical shifts) and MS (through cross-links) proved highly complementary and mutually supportive and allowed to build models for the two complexes using computational docking. In a second step, we used the obtained models to find small molecule scaffolds that can bind to either of the complex members in their interaction regions as determined from the integrative models (fragment screening). Using a small, fluorine-labeled compound library, low-affinity binders were discovered for all targets (N-NTD, N-CTD, s2m) using 19F-NMR. Such compounds could be further optimized using lead optimization strategies, although this was not attempted here. Our integrative workflow highlights the combination of different experimental techniques, including cross-linking MS, for the structural characterization of protein-RNA complexes. Beyond the illustrated example with a potential application in drug discovery, we are currently also exploring the value of this approach for different applications in biophysics and systems biology.

Novel aspect: Combination of protein-RNA cross-linking data with other experimental and computational methods in a drug discovery context

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