Unwinding the helicase NSP13

Background

SARS-CoV-2 belongs to the genus Betacoronavirus and has an approximately 30 kb long positive-sense single-stranded RNA genome. It codes for various structural and non-structural proteins, including helicase. Helicases are nucleotide-dependent nucleic acid unwinding enzymes that are vital during the virus life cycle. SARS encodes its own helicase, which is NSP13. It utilises energy from nucleotide hydrolysis to unwind the substrates (nucleic acids) and translocate on the strand. Motor protein NSP13 shows a directionality bias. It moves from 5’ to 3’ direction along a strand.

Domain architecture of NSP13

Organisation of domains of NSP13

NSP13 is a motor protein having 601 amino acids. It comprises five different domains. Starting from the amino terminus, it has a (ZBD) (1-100), followed by a containing three helices (101-150), a having a beta-barrel (151-261), (262-442) and (443-601).

Functions and structural architecture of domains of NSP13

Zinc binding domain

The zinc-binding domain is unique to the order Nidovirales. Zinc zinc-binding domain coordinates three zinc ions through structural Zn2+ binding clusters, which contain conserved cysteine residues. The first zinc cluster has a Cys-Cys-Cys-Cys (CCCC) configuration. The second cluster has Cys-Cys-His-His (CCHH) configuration, and the third zinc ion is coordinated by Cys-Cys-Cys-His (CCCH) finger. The zinc-binding domain is also known to interact with the N-terminus helical region of NSP8.

Stalk

The stalk domain is made up of three helices. Two of them are comparatively shorter than the third one. The stalk domain connects the zinc binding domain to the 1B domain. It is suggested that there could be a signal relay mechanism between the zinc-binding domain and the helicase domain, which is transmitted by a stalk domain.

1B domain

The 1B domain consists of a six-anti-parallel β-barrel. A 30 amino acid-long linker connects the β barrel to the helicase subdomains.

RecA like helicase subdomain 1A

1A domain consists of seven parallel β sheets sandwiched between five alpha helices. Two of these helices are on the stalk domain side, and three are on the opposite side. 1A domain is connected to the 2A domain via a short five-amino acid containing hinge region. Four conserved helicase motifs like motif I, Ia, II and III can be found in the 1A domain.

RecA like helicase subdomain 2A

2A domain has a five parallel beta sheet structure surrounded by six alpha helices. Together with the 1A domain, they make up a core helicase domain with conserved motifs. Motif IV, V and VI, which are helicase conserved motifs. These motifs facilitate nucleotide binding.

Classification of helicases based on superfamilies

Helicases are mainly classified into six superfamilies from SF1 to SF6. This classification is based on the conserved sequence motifs and structural similarities. These families are also categorised into two main groups based on their oligomeric state.

Non ring forming helicases

Typically include SF1 and SF2. They function as monomers or dimers and are involved in cellular processes.

Ring forming helicases

Include SF3 to SF6. They form hexameric or multimeric rings around the DNA and include the replicative helicases.

Superfamily 1 (SF1)

SF1 helicases are the most characterised family. Based on the direction of translocation, they are further classified as type A and type B.

SF1A Helicases

In these helicases, the direction of translocation is from 3’ to 5’ polarity. The best studied enzymes in this class include Rep, UvrD and PcrA from the bacteria. Structural studies have shown that they have an ATP binding site, which is located in a cleft between the two RecA-like subdomains called as N and C core.

SF1B Helicases

In these helicases, the direction of translocation is from 5’ to 3’. Examples include RecD (RecBCD complex) and Dda helicases from bacteria. Eukaryotic members are Pif1 and Rrm3. SF1B enzymes have two RecA-like core domains with the ATP binding site the same as SF1A, but they have an additional N-terminal domain.

Superfamily 2 (SF2)

SF2 enzymes are the largest superfamilies. They include DEAD box RNA helicases, RecQ-like enzymes and Snf2-like helicases. Majorly, SF2 family members display 3’ to 5’ polarity.

Superfamily 3 (SF3)

Members of this family form hexamers or double hexamers. They have a directionality from 3’ to 5’. One of the helicases from this family is the E1 helicase from papillomavirus.

Superfamily 4 (SF4)

All characterised members from the family have 5’ to 3’ polarity. One of the most studied examples from the family includes gene 4 protein (gp4) from T7 bacteriophage.

Superfamily 5 (SF5)

One of the members from SF5 is Rho. Rho is required for the termination of transcription in bacteria and is responsible for unwinding the DNA/RNA hybrid.

Superfamily 6 (SF6)

This family mainly contains hexameric motor proteins having the AAA+ fold. One such example from eukaryotic helicases is the mini chromosome maintenance (MCM) protein. RuvB protein from prokaryotes also falls in this family.

Mechanism of unwinding of nucleic acids and translocation by helicases

During the unwinding process, the RNA binds between the 1A and 1B domains with the 5’ end held between the cleft between these two domains and the 1B domain. The conserved motif between the two Rec1A and Rec1B domains forms the key contacts. Motif I and II have classical walker A and walker B motifs. Motifs I, II and IV mainly bind to ATP, while motif IV exclusively binds to RNA and motifs Ia, V and III bind to both ATP and RNA. Structural data from the structure of AMP-PNP with NSP13 shows that the adenine base makes stacking interactions with H290 and R442. The O3 ribose makes a hydrogen bond with E540, which is part of motif V. The α phosphate forms a salt bridge interaction with the main chain of H290 and the side chain of K320. The β phosphate is situated within the phosphate-binding motif I. The γ phosphate makes extensive interactions with the side chains of Q404, R443 and R567, which are from motifs III, IV and VI, respectively. The Mg2+ ion is in coordination and makes contact with β and γ phosphate. With our current understanding of NSP13, the suggested model of translocation is by inchworming. In this model, ATP hydrolysis drives the alternating formation of contacts in the separate RecA-like domains to take turns grabbing and releasing nucleic acids. ATP binding causes contacts with motifs IV, V and VI from Rec2A to slide along the nucleotide chain. Rec1A motif III interacts with motif Ia and generates a pocket around the nucleotide and towards the base. After one hydrolysis cycle, the structure relaxes, coming to its original conformation. In summary, when ATP binds to the NSP13-dsRNA complex, the 1A and 2A domains come together, and the 2A domain binds more tightly to the RNA while 1A relaxes and is stabilised by the 1B domain. During the transition state, the 1A domain slides along the RNA while moving away from the 2A domain. In the product state, 1A and 2A continue to move away from one another. 1A continues to tighten to bind with the nucleic acid.