Revealing SARS-CoV-2 protein architectures through in-situ MS proteomics and integrative modeling
Creators: name: Linial, Michal
name: Schneidman-Duhovny, Dina
name: Kalisman, Nir
Publisher: name: F1000 Research Limited
Year: 2021

description: <b>Revealing SARS-CoV-2 protein architectures through in-situ MS proteomics and integrative modeling </b><br> Michal Linial<sup>1</sup>, Dina Schneidman-Duhovny<sup>1,2</sup>, Nir Kalisman<sup>1</sup><br> <br> <sup>c</sup>Department of Biological Chemistry, Institute of Life Sciences, <sup>1,2</sup>The Rachel and Selim Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel<br> <br> <b>Short Abstract</b><br> The genome of SARS-CoV-2, the causal virus of the COVID-19 pandemic, encodes 29 proteins. However, only a handful of them is associated with structure and function. In this study, we utilize a novel application called in situ cross-linking mass spectrometry (in situ CLMS) that provides rich spatial information on the structures of proteins as they occur in intact cells. We demonstrate the utility of this approach by targeting three SARS-CoV-2 proteins for which full atomic structures are missing. We show that integrating cross-links with external structural data is sufficient to model the full-length protein. Cells that expressed tagged-Nsp1 were subjected to in situ CLMS approach. We identified the interactions of Nsp1 with the 40S ribosomal subunit which confirms its fundamental role in blocking the translation of infected cells. Similarly, based on structure predictions of individual domains for Nsp2 by AlphaFold2, we successfully assembled Nsp2 into a single consistent model. The Nucleocapsid (N) protein plays a key role in genome packing and virion assembly. Using in situ CLMS was fundamental to assemble a model of the full dimer from available 3D structures of individual domains. These results highlight the importance of cellular context for achieving detailed atomic resolution of SARS-CoV-2 proteins.<br> <br> <b>Introduction</b><br> The approach of <i>In situ cross-linking mass spectrometry</i> (in situ CLMS) allows the probing of protein structure inside intact cells. In this approach, cells are incubated with a membrane-permeable cross-linking reagent, which reacts with the proteins in their native environment. The cells are lysed and their protein content is analyzed by mass spectrometry (MS) following the chemical cross-linking. Mass search can identify pairs of covalently linked residues, providing a rich resource for modeling protein structures and interactions [1].<br> To reduce the complexity before the MS analysis, tagged proteins are used which allows purifying the protein-of-interest out of the cell lysate before its preparation for the MS. In this work, we introduce a modified protocol to improve the purification steps and applied it to probe three SARS-CoV-2 proteins. These proteins suffer from incomplete knowledge and only partial structural information. Thus, we used these proteins for assessing the feasibility of the method to provide reliable structural models. We focused on three SARS-CoV-2 proteins for which the structural information is either missing or incomplete: Nsp1 (180aa), Nsp2 (639aa), and the Nucleocapsid protein (N protein, 419aa). The expression levels of all three proteins were optimized before the application of the in situ CLMS.<br> <br> <b>Methods</b><br> We employed a targeted strategy for <i>in situ</i> CLMS of viral proteins inside intact human cells. To that end, HEK293 cells were transfected with a plasmid of a selected viral protein fused to a Strep-tag. We then cross-linked the intact cells with a membrane-permeable cross-linker (either DSS or formaldehyde), washed away the excess cross-linker, lysed the cells, and purified the viral protein via the Strep-tag. The purification step greatly enriches and simplifies the sample<b> [2]</b>.<br> <br> <b>Results</b><br> <br> We illustrate the success of providing an accurate atomic model for <b>Nsp2</b>. There are no close homologs with a solved structure available for Nsp2. We have therefore referred to the Nsp2 model generated by AlphaFold2 from DeepMind. AlphaFold2 has been highly successful in the recent CASP14 round, submitting highly accurate models. Notably, the initial AlphaFold2 model violated many of the cross-links (39%). A cross-link is considered violated if the Cɑ-Cɑ distance is higher than 25 Å. Importantly, the violated cross-links were mostly inter-domain, while almost all intra-domain cross-links were satisfied. We divided the AlphaFold2 model into its individual six domains to obtain a model consistent with the cross-link set. One domain that was not covered by the initial AlphaFold2 model, was modeled by homology to partial Nsp2 structure of the infectious bronchitis virus (PDB 3ld1, sequence identity 13%). With the availability of the structures for the individual domains, the modeling task is converted into a domain assembly problem. The six domains served as an input along with the primary set of cross-links and domain connectivity constraints. We have obtained 62 models that satisfied all the primary set inter-domain cross-links with a precision of 8 Å <b>[3]</b>. These models converged into a single cluster with a higher precision of 1 Å.<br> <br> <b>Structural model of </b><b>Nucleocapsid </b><b>N-protein. </b>Another challenging case is the structural modeling of <b>N-protein</b>. The main difficulty is that the N protein in the cell-infected context is a master gene for RNA binding and virion assembly. In our overexpressed N protein, viral RNA is not accessible. Still, of special interest are cross-links that contain an overlapping peptide pair. Such cross-links necessarily report on direct interaction between two chains of N. We identified four sequence regions that form such cross-links. These identifications are supported by well-annotated MS/MS fragmentation spectra. In several cases, these identifications are also supported by more than one peptide pair. The dimer model can explain the inter-chain cross-links between Lys100 and Lys102 that are closer than 25 Å in several docking solutions.<br> <br> <b>Conclusions</b><br> We present a generic methodology that extracts structural data from living, intact cells for any protein-of-interest. Application of this methodology to different viral proteins resulted in significant cross-link sets that revealed their internal structural connectivity. Importantly, we show that these cross-link sets are detailed enough to enable the integrative modeling of the full-length protein sequence. Consequently, we confirmed the Nsp1 role as a translation initiation blocker. In this work, we provide results for the first global structural organization of Nsp2 and the dimer of the N protein. We foresee that similar applications will be highly useful to study other recalcitrant proteins on which the mainstream structural approaches currently fail.<br> <br> <b>The research team [4] </b>(Hebrew University): Moriya Slavin<sup>§</sup>, Joanna Zamel<sup>§</sup>, Keren Zohar<sup>§</sup>, Siona Eliyahu, Merav Braitbard, Esther Brielle, Leah Baraz, Miri Stolovich-Rain, Ahuva Friedman, Dana G Wolf, Alexander Rouvinski, Michal Linial<sup>*</sup>, Dina Schneidman-Duhovny<sup>*</sup>, Nir Kalisman<sup>*</sup><br> <sup>§</sup>These authors contributed equally; <sup>*</sup>Corresponding authors<br> <br> <b>References</b><br> 1. Leitner, A. Bonvin, A.M. Borchers, C.H. et al. (2020) Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry. Structure, 28, 1259-1268.<br> 2. Tayri-Wilk, T. Slavin, M.; Zamel, J. et al. (2020) Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins. Nature communications 11, 1-9.<br> 3. Schneidman-Duhovny, D. Wolfson, H. J. (2020) Modeling of Multimolecular Complexes. Methods Mol. Biol. 2112, 163–174.<br> 4. Slavin, M. Zamel, J., Zohar, K. et al. (2021) Targeted <i>in situ </i>cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2<b>. </b>PNAS<b> </b>(in press).