Role of Mutations in Differential Recognition of Viral RNA Molecules by Peptides

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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.

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Pal

Kumar

Vashisth

2023

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“…We have reported the uncertainities in the averaged Δ G comp and Δ G free values as the standard error of the mean and have calculated the error in the final ΔΔ G by propagating the standard error of the mean associated with the averaged Δ G comp and Δ G free values. For each calculation, we obtained a convergence of the computed ΔΔ G with a reasonable statistical uncertainty (<1 kcal/mol). ,,− We report the free energy calculations with an aggregate time of ∼1.89 μs.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, it is of significance to probe the dynamics and the thermodynamic basis of ligand binding in the NMT1 riboswitch for a broader understanding of the role of riboswitches as potential antibacterial drug targets and for identifying novel riboswitch-targeting compounds. Here, MD simulations − and the structure-based free energy calculation methods are useful tools to probe the conformational dynamics and the ligand recognition mechanism of RNA influenced by mutations. , These computational methods have been extensively adopted to explore the interaction mechanisms of biomolecules with small organic ligands with sufficiently high accuracy. − …”

Section: Introductionmentioning

confidence: 99%

Mechanism of Ligand Discrimination by the NMT1 Riboswitch

Kumar

Vashisth

2023

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Riboswitches are conserved functional domains in mRNA that almost exclusively exist in bacteria. They regulate the biosynthesis and transport of amino acids and essential metabolites such as coenzymes, nucleobases, and their derivatives by specifically binding small molecules. Due to their ability to precisely discriminate between different cognate molecules as well as their common existence in bacteria, riboswitches have become potential antibacterial drug targets that could deliver urgently needed antibiotics with novel mechanisms of action. In this work, we report the recognition mechanisms of four oxidization products (XAN, AZA, UAC, and HPA) generated during purine degradation by an RNA motif termed the NMT1 riboswitch. Specifically, we investigated the physical interactions between the riboswitch and the oxidized metabolites by computing the changes in the free energy on mutating key nucleobases in the ligand binding pocket of the riboswitch. We discovered that the electrostatic interactions are central to ligand discrimination by this riboswitch. The relative binding free energies of the mutations further indicated that some of the mutations can also strengthen the binding affinities of the ligands (AZA, UAC, and HPA). These mechanistic details are also potentially relevant in the design of novel compounds targeting riboswitches.

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

Kumar,

Vashisth

2024

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The highly conserved protease enzyme from SARS-CoV-2 (M Pro ) is crucial for viral replication and is an attractive target for the design of novel inhibitory compounds. M Pro is known to be conformationally flexible and has been stabilized in an extended conformation in a complex with a novel nanobody (NB2B4), which inhibits the dimerization of the enzyme via binding to an allosteric site. However, the energetic contributions of the nanobody residues stabilizing the M Pro /nanobody interface remain unresolved. We probed these residues using all-atom MD simulations in combination with alchemical free energy calculations by studying the physical residue−residue interactions and discovered the role of hydrophobic and electrostatic interactions in stabilizing the complex. Specifically, we found via mutational analysis that three interfacial nanobody residues (Y59, R106, and L109) contributed significantly, two residues (L107 and P110) contributed moderately, and two residues (H112 and T113) contributed minimally to the overall binding affinity of the nanobody. We also discovered that the nanobody affinity could be enhanced via a charge-reversal mutation (D62R) that alters the local interfacial electrostatic environment of this residue in the complex. These findings are potentially useful in designing novel synthetic nanobodies as allosteric inhibitors of M Pro .