Deducing the Crystal Structure of MERS-CoV Helicase

Abstract

RNA virus encodes a helicase essential for viral RNA transcription and replication when the genome size is larger than 7 kb. Coronavirus (CoV) has an exceptionally large RNA genome (~30 kb) and it encodes an essential replicase, the nonstructural protein 13 (nsp13), a member of superfamily 1 helicases. Nsp13 is among the evolutionary most conserved proteins not only in CoVs but also in nidovirales. Thus, it is considered as an important drug target. However, the high-resolution structure of CoV nsp13 remained unavailable even until more than a decade after the outbreak of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, which hindered the structure-based drug design. This is in part due to the intrinsic flexibility of nsp13. Here, we describe protocols of deducing the crystal structure of Middle East respiratory syndrome coronavirus (MERS-CoV) helicase in detail, which include protein expression, purification, crystallization, enzymatic characterization, and structure determination. With these methods, catalytically active recombinant MERS-CoV nsp13 protein can be prepared and crystallized and the crystal structure can be solved.

State-of-the-art tools unveil potent drug targets amongst clinically approved drugs to inhibit helicase in SARS-CoV-2

“…Helicase enzyme in coronavirus is a prominent viral replication enzyme. Helicases are evolutionarily conserved proteins in coronaviruses and Nidovirales [27]. Furthermore, double-stranded nucleic acids are separated into two single-stranded nucleic acids by helicases, which catalyse the separation [10].…”

Analysis of the SARS-CoV-2-host protein interaction network reveals new biology and drug candidates: focus on the spike surface glycoprotein and RNA polymerase

“…Moreover, interaction with epigenetic, gene regulatory pathways and cytoskeleton was shown [ 3 ]. The crystal structure was resolved recently for the MERS-CoV virus [ 110 ] and study of the crystal composed of the SARS-CoV-2 holo-RdRP and NSP13 confirmed that a stable complex could be formed [ 98 ]. The interaction of the two proteins is thought to be mediated by the ZBD domain of SARS-CoV-2 NSP13 and the extension of the NSP8 subunit.…”

Mechanism of duplex unwinding by coronavirus nsp13 helicases

“…As a member of the SF1B helicase family, nsp13 unwinds RNA or DNA duplexes with 5'-to-3' directionality and hydrolyzes both NTP and dNTP (7,8); it also hosts 5'triphosphatase activity. In previous work, by determining the crystal structure of MERS-CoV nsp13 (PDB ID: 5WWP), we revealed that nsp13 generally mirrors the domain organization of nidovirus helicases, whereas the individual domains (i.e., CH, RecA1-1B and RecA2) of nsp13 reflect the organization of cellular Upf1-like helicases (9,10).…”