Role of Dynamics and Mutations in Interactions of a Zinc Finger Antiviral Protein with CG-rich Viral RNA

Abstract

Zinc finger antiviral protein (ZAP) is a host antiviral factor that selectively inhibits the replication of a variety of viruses. ZAP recognizes the CG-enriched RNA sequences and activates the viral RNA degradation machinery. In this work, we investigated the dynamics of a ZAP/RNA complex and computed the energetics of mutations in ZAP that affect its binding to the viral RNA. The crystal structure of a mouse-ZAP/RNA complex showed that RNA interacts with the zinc finger 2 (ZF2) and ZF3 domains. However, we found that due to the dynamic behavior of the single-stranded RNA, the terminal nucleotides C1 and G2 of RNA change their positions from the ZF3 to the ZF1 domain. Moreover, the electrostatic interactions between the zinc ions and the viral RNA provide further stability to the ZAP/RNA complex. We also provide structural and thermodynamic evidence for seven residue pairs (C1–Arg74, C1–Arg179, G2–Arg74, U3–Lys76, C4–Lys76, G5–Arg95, and U6–Glu204) that show favorable ZAP/RNA interactions, although these interactions were not observed in the ZAP/RNA crystal structure. Consistent with the observations from the mouse-ZAP/RNA crystal structure, we found that four residue pairs (C4–Lys89, C4–Leu90, C4–Tyr108, and G5–Lys107) maintained stable interactions in MD simulations. Based on experimental mutagenesis studies and our residue-level interaction analysis, we chose seven residues (Arg74, Lys76, Lys89, Arg95, Lys107, Tyr108, and Arg179) for individual alanine mutations. In addition, we studied mutations in those residues that are only observed in the crystal structures as interacting with RNA (Tyr98, Glu148, and Arg170). Out of these 10 mutations, we found that the Ala mutation in each of the five residues Arg74, Lys76, Lys89, Lys107, and Glu148 significantly reduced the binding affinity of ZAP to RNA.

Adenine Methylation Enhances the Conformational Flexibility of an RNA Hairpin Tetraloop

“… 80 By utilizing the state-function property of the free energy, the final free-energy difference can be computed by integrating over free-energy values in each intermediate state. This technique has been successfully applied to determine the energetic contribution of individual residues in RNA/protein, 65 , 66 , 68 protein/protein, 81 and protein/ligand 82 systems.…”

“…In this work, we applied all-atom MD simulations with alchemical free-energy calculations − to probe the effects of m 6 A modification on the RNA structure at two distinct structural levels. Initially, we characterized the energetics of syn / anti configurations adopted by the methyl group in the methylated adenine mononucleotide, thus resolving the effects of methylation on one of the basic building blocks of RNA .…”

Synthesis and Antimicrobial Applications of ZnO Nanostructures: A Review

“…It is noteworthy that most of the papers available in literature deal with the use of Zn 2+ ions as antiviral agents, rather than ZnO NSs, possibly in combination with other molecules like lactoferrin, heparin, etc. − Zinc ions can penetrate cell walls (especially when driven by ionophores), augmenting the intracellular Zn 2+ concentration and subsequently impairing virus replication . Bhatti and DeLong recently reviewed the antiviral application of different nanomaterials, including ZnO NSs: in that paper, ROS generated through photocatalytic reactions driven by UV–Vis light on ZnO NSs are indicated as the main species responsible for damaging of the viral envelopes, interfering with viral entry and replication …”

Quantitative Assessment of Energetic Contributions of Residues in a SARS-CoV-2 Viral Enzyme/Nanobody Interface

“…The residue-specific free energy changes were quantified using the free energy perturbation (FEP) method which was applied using an appropriate thermodynamic cycle (Figure S2). − Overall, we performed an aggregate of ∼24 μs of all-atom MD simulations and free energy calculations to provide the structural and energetic basis for the recognition of M Pro by the nanobody. We also suggest mutations that can potentially enhance the affinity of the nanobody for M Pro .…”