Targetedin situcross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

Abstract

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.

The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex

“…At the structural level, NSP2 has a complex multi-domain topology including an N-terminal domain with a highly conserved zinc binding site, and a C-terminal region rich in b-strands. With the exception of the zinc binding site, NSP2 displays a rapidly evolving surface with the presence of natural variations that could impact host-virus interactions (Ma et al, 2021;Mompean et al, 2021;Gupta et al, 2021;Slavin et al, 2021). Among its host interactors, SARS-CoV-2 NSP2 interacts with the 4EHP-GIGYF2 complex, a key machinery in translational silencing and mRNA decay (Davies et al, 2020;Cornillez-Ty et al, 2009;Gordon et al, 2020aGordon et al, , 2020b.…”

In-Cell Labeling and Mass Spectrometry for Systems-Level Structural Biology

“…A study focusing on three proteins of the SARS-CoV-2 virus (Nsp1, Nsp2 and nucleocapsid) expressed strep tagged versions of the viral proteins in HEK293 cells prior to cross-linking the cells with DSS . The cells were lysed and the viral proteins were purified via the strep tag prior to XL-MS analysis.…”

“…The cells were lysed and the viral proteins were purified via the strep tag prior to XL-MS analysis. Identified cross-linked residues served as distance restraints for integrative modeling and domain assembly of Nsp2, olgimerization and domain assembly of nucleocapsid, and complex assembly between the viral protein Nsp1 and the host ribosomal protein RS3 . Importantly the authors found that the in situ cross-linking results were not fully replicated by in vitro experiments indicating protein states that are unique to the cellular environment …”

“…107 A study focusing on three proteins of the SARS-CoV-2 virus (Nsp1, Nsp2 and nucleocapsid) expressed strep tagged versions of the viral proteins in HEK293 cells prior to cross-linking the cells with DSS. 108 The cells were lysed and the viral proteins were purified via the strep tag prior to XL-MS analysis. Identified cross-linked residues served as distance restraints for integrative modeling and domain assembly of Nsp2, olgimerization and domain assembly of nucleocapsid, and complex assembly between the viral protein Nsp1 and the host ribosomal protein RS3.…”

Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

“…The N-terminal domain could be located in the cryo-EM maps but at too low a resolution to allow model building, presumably due to flexibility with respect to the 40S. The cryo-EM derived location of the Nsp1 N-terminal domain in the ribosomal complex is further supported by in situ cross-linking mass spectrometry identifying multiple crosslinks between Nsp1 and ribosomal protein S3 (18,20).…”