The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen successfully uses multiple immune evasion mechanisms to achieve infection within its host. An intriguing new study, which was released on the bioRxiv* preprint server, describes one such process, which may help develop drugs to counteract the virus more effectively.
RNA capping is a term used to denote the enzymatic modification of the 5’ end of the RNA viral genome. This is crucial to the efficient synthesis of viral proteins, prevention of viral RNA breakdown by host RNases, and immune evasion.
In coronaviruses, this process terminates with the assembly of the nonstructural protein 16 (nsp16), and its non-catalytic stimulator nsp10 on the 5’ end of the new growing RNA strand to perform S-adenosyl-L-methionine (SAM)-dependent methylation of the 2’-OH on the first nucleotide (N1), to modify the RNA cap.
This step converts the RNA from Cap-0 to Cap-1. As a result, the innate immune response to the virus is suppressed.
The current study uses a molecular replacement method to explore the structure of the nsp16/nsp10 heterodimer complex with Cap-1. The Cap-1 includes N1, the adjacent N2, and a methylation byproduct, SAH (S-adenosyl-L-homocysteine).
Breathing motion of the enzyme
The researchers found that the nsp16/nsp10 complex showed expansion compared to the substrate-bound enzyme. Whereas the nsp16 bound to the substrate Cap-0 showed the canonical structure of a central β-sheet with two α-helices on one side and three on the other, in the nsp16/nsp10 complex, various shifts and rotations occur.
The conformation of the SAH-bound enzyme thus changes in order to facilitate its ‘resetting’ for renewed catalytic activity in the next step. The heterodimer shows a widened interface, with increased relaxation. This is the result of a single 2’-O methylation event.
While catalytic activity is occurring, the enzyme shows “breathing motion,” that is, the substrate, product and byproduct bound states are just the fully closed, open and partly open states of the enzyme. The Cap-1 is fitted into a deep pocket between the central β-sheet and the two gate loops. The SAH byproduct fits into a cavity at the C-terminal side of the parallel β-strands. These two differ only in the orientations of their carboxy termini.
Structures of SARS-CoV-2 nsp16/nsp10 complexes. a, The substrate (me7GpppA, blue stick) and methyl donor (SAM, yellow stick)-bound nsp16 (cyan)/nsp10 (orange) complex (PDB ID, 6WKS)8 represents a closed form. b, The product (me7GpppAmU, red stick; byproduct SAH [grey stick])-bound nsp16 (blue)/nsp10 (magenta) in an open state. A yellow circle shows the methylated ribose (2’-O-me) of N1 (A) base. c, The SAH (grey) bound nsp16 (grey)/nsp10 (pink) represents a partially open or enzyme reset state. d, Secondary structure-based overlay of nsp16 in substrate- and product-bound states clearly shows the universal expansion of the enzyme upon 2’- O methylation. e, A close up view of Cap-binding and catalytic pocket of the product structure shows nsp16 residues (cyan sticks) interacting with Cap-1 (red). A positional change in orientation of the substrate (Cap-0, blue) from the “closed” structure determined previously8 is shown. f, An overlay of the product (Cap-1) and byproduct (SAH)-bound structures shows change in the orientation of gate loop 2. Reduction in buried surface area between nsp16/nsp10 in fully and partially open structures (compared to substrate-bound closed state) is shown (g-i).
Metal ion plays a crucial role
The study also shows that a divalent metal ion interacts with the water molecules during 2’-O MTase activity. Metal ions stabilize nucleic acid substrates as well as catalyze enzymatic reactions, such as magnesium ions in the dengue virus.
The wildtype nsp16/nsp10 heterodimer binds magnesium with high affinity, allowing direct metal-protein interaction. This is found only in SARS-CoV-2, as is its orientation in the same binding pocket as Cap-1. It directly binds to nsp16 and to the phosphate of the uridine second nucleotide.
With dengue, conversely, the magnesium ion does not ligate the protein while crosslinking the RNA cap phosphate groups.
Positioning within catalytic pocket
The researchers also demonstrated the role of K46, K170, and N198 in catalysis, through their network of side chains. This is important to achieve the right positioning of the RNA cap within the catalytic pocket. This ensures specific 2’-O methylation of the first nucleotide.
By preventing its movement or wrong positioning during this step, this network also prevents the inadvertent 2’-O methylation of the second.
Mutations in clinical variant
They also explored the mutations of the S33 residue in gate loop 1, which was involved in the New York City epidemic as well as in other previous coronavirus outbreaks. The side chains of this amino acid may interfere with magnesium binding in the catalytic pocket, thus misaligning the first nucleotide.
Instead, a shorter side chain would not only avoid such intrusion but could yield more contacts with the divalent metal ion, provide stronger RNA binding and thus facilitate the reaction.
They found that N198 and K46 totally wiped out nsp16 catalytic activity, while S33R mutation showed an 80% reduction in activity. However, the S33N mutation increased the activity by 30%.
Impact of metal substitution
When calcium is substituted for magnesium, the wildtype nsp16 shows a 20% loss, but not for manganese. With the S33N mutant, all three divalent ions performed comparably. The S33R showed 80% less activity with manganese and magnesium, relative to the wildtype enzyme, but residual activity with calcium.
The divalent metal does not appear to play a chemical role in the SARS-CoV-2 nsp16, but its key place in the 2’-O methylation of N1 is plain. As a result, an alteration in the cellular metal ion concentrations could change the progress of RNA capping.
What are the implications?
The study suggests that the conformational changes in the enzyme refresh it for repeated rounds of catalysis. These include widening on product formation, and an inward twisting of the substrate binding region upon releasing the product.
The researchers also found a role for divalent metals with a unique direct metal-protein binding mode that appears to be essential for the 2’-O methylation of N1.
A clinical variant of the virus shows altered enzymatic activity, for which a structural explanation was found as well.
The outcome of low RNA capping due to low metal concentrations could be reduced host immune response evasion. For instance, low calcium and low magnesium levels serve as predictors of in-hospital deaths in COVID-19 patients, and are commonly found in severe COVID-19, respectively.
This could be due to low RNA capping, which, along with the low levels of divalent metal ions, causes hyperinflammatory responses in some COVID-19 patients.
Further research will show if this hypothesis is valid, namely, by exploring how RNA capping is related to metal levels in the host cell and to the innate immune response.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.