An iron-sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities

Nunziata Maio; Md Kausar Raza; Yan Li; De-Liang Zhang; J Martin Bollinger Jr; Carsten Krebs; Tracey A Rouault

doi:10.1073/pnas.2303860120

An iron-sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities

Proc Natl Acad Sci U S A. 2023 Aug 15;120(33):e2303860120. doi: 10.1073/pnas.2303860120. Epub 2023 Aug 8.

Authors

Nunziata Maio¹, Md Kausar Raza², Yan Li³, De-Liang Zhang¹, J Martin Bollinger Jr^{2

4}, Carsten Krebs^{2

4}, Tracey A Rouault¹

Affiliations

¹ Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892.
² Department of Chemistry, The Pennsylvania State University, University Park, PA 16802.
³ National Institute of Neurological Disorders and Stroke, NIH, Proteomics Core Facility, Bethesda, MD 20892.
⁴ Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an Fe₄S₄ cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.

Keywords: SARS-CoV-2; iron-sulfur clusters; nsp13; viral helicase.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

COVID-19 Drug Treatment
COVID-19*
DNA Helicases / metabolism
Humans
RNA
RNA Helicases / genetics
SARS-CoV-2* / genetics
Sulfur
Viral Nonstructural Proteins / metabolism

Substances

DNA Helicases
RNA
Sulfur
Viral Nonstructural Proteins
RNA Helicases