PT  - JOURNAL ARTICLE
AU  - Moriya Slavin
AU  - Joanna Zamel
AU  - Keren Zohar
AU  - Siona Eliyahu
AU  - Merav Braitbard
AU  - Esther Brielle
AU  - Leah Baraz
AU  - Miri Stolovich-Rain
AU  - Ahuva Friedman
AU  - Dana G Wolf
AU  - Alexander Rouvinski
AU  - Michal Linial
AU  - Dina Schneidman-Duhovny
AU  - Nir Kalisman
TI  - Targeted <em>in situ</em> cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2
AID  - 10.1101/2021.02.04.429751
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.04.429751
4099  - http://biorxiv.org/content/early/2021/02/04/2021.02.04.429751.short
4100  - http://biorxiv.org/content/early/2021/02/04/2021.02.04.429751.full
AB  - Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.Competing Interest StatementThe authors have declared no competing interest.