Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region

Abstract

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many viruses use their own IDPs to “hack” these processes to deactivate host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational preferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2, whose C-terminal region (CtR) is predicted to be disordered, interacts with human proteins that regulate translation initiation and endosome vesicle sorting. Molecules that block these interactions could be valuable leads for drug development. The 13Cβ and backbone 13CO, 1HN, 13Cα, and 15N nuclei of Nsp2’s 45-residue CtR were assigned and used to characterize its structure and dynamics in three contexts; namely: (1) retaining an N-terminal His tag, (2) without the His tag and with an adventitious internal cleavage, and (3) lacking both the His tag and the internal cleavage. Two five-residue segments adopting a minor extended population were identified. Overall, the dynamic behavior is midway between a completely rigid and a fully flexible chain. Whereas the presence of an N-terminal His tag and internal cleavage stiffen and loosen, respectively, neighboring residues, they do not affect the tendency of two regions to populate extended conformations.

SARS-CoV-2 NSP2 as a Potential Delivery Vehicle for Proteins

“…19,20 Existence of intrinsically disordered regions (IDRs) within the C-terminal region of SARS-CoV-2 NSP2 was also determined by using NMR spectroscopy. 16 We found that NSP2 of SARS-CoV-2 can be secreted into the culture supernatants of transfected cells. In addition, NSP2 protein from different coronaviruses, such as SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), or porcine epidemic diarrhea virus (PEDV), was also secreted by the cells.…”

“…14,15,22 Similar to the Tat protein of HIV-1, SARS-CoV-2 NSP2 contains IDRs. [15][16][17]19,20,23 We then investigated whether the SARS-CoV-2 NSP2 protein could be secreted as a Tat protein. Expression vectors of SARS-CoV-2 NSP2, NSP5, and NSP8 were transfected into HEK293T cells.…”

Intrinsically Disordered Proteins: Perspective on COVID-19 Infection and Drug Discovery

“… 6 The C-terminal region of the nonstructural protein Nsp2 has been predicted to be disordered. 107 Because it interacts with host proteins, which regulate translation initiation and endosome vesicle sorting, compounds that block these interactions could be valuable candidates for drug development. Although the other proteins exhibit lower disorder content, nearly all contain at least one IDR.…”

“…In the group of the nonstructural SARS-CoV-2 proteins, several moderately disordered proteinsNsp8, ORF6, and ORF9bhave been identified, as well . The C-terminal region of the nonstructural protein Nsp2 has been predicted to be disordered . Because it interacts with host proteins, which regulate translation initiation and endosome vesicle sorting, compounds that block these interactions could be valuable candidates for drug development.…”

COVID-19 therapies: do we see substantial progress?

“…nsp2 , a 638-amino-acid-residue protein, was found to interact with host-cell proteins regulating initiation of translation and endosome vesicle sorting. It is interesting that its C-terminal domain, which is disordered, plays a crucial role in these interactions [ 19 ].…”