Atomic resolution structures of viral protein assemblies by magic angle spinning NMR spectroscopy and density functional theory calculations for accurate determination of NMR parameters
Date
2022
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Publisher
University of Delaware
Abstract
Structure determination of pathogenic virus particles at atomic resolution is essential for understanding the biological processes pertaining to the virus. A high resolution structural information of viral protein significantly facilitates therapeutic discovery for combating the disease pathogenesis. Nuclear magnetic resonance (NMR) spectroscopy is a unique technique providing atomic resolution details on structures and dynamics of viral protein assemblies. This dissertation mainly focuses on the two applications of biomolecular NMR. In the first part, we utilized solid-state magic angle spinning (MAS) NMR to determine structures of two functionally important viral protein assemblies, an HIV-1 Gag polyprotein maturation intermediate and the SARS-CoV-2 nucleocapsid N-terminal domain protein. In NMR, chemical shift is the measurable quantitative value which is sensitive towards local electronic environment around the nucleus of interest. Thus, it is a key player in structure determination of a variety of molecules. Quantum mechanical calculation of NMR parameters gives insight into important structural information and can be integrated with experimental NMR to characterize molecular structures. In the second part of the dissertation, I discuss how we apply Density Functional Theory (DFT) for accurate quantum chemical calculations of NMR parameters of pharmaceutical compounds and HIV-1 capsid protein cluster models. Chapter 1 presents a general overview of methodologies for determination of molecular structure of viral proteins by solid-state MAS NMR. ☐ Chapter 2 presents elucidation of the structural basis of an HIV-1 maturation inhibitor binding and activity to an immature HIV-1 Gag polyprotein fragment. HIV-1 contains an assembly and maturation switch, which spans the C-terminal domain (CTD) of the capsid (CA) region and the first spacer peptide (SP1) of the Gag polyprotein. The HIV-1 maturation inhibitors (for example, Bevirimat or BVM, PF-46396, and their analogs), which have emerged as a novel class of antiretroviral treatments, disrupt the final step of protease-mediated Gag processing by binding to the CACTD-SP1 junction of the immature Gag lattice. The binding of maturation inhibitors to the CACTD-SP1 junction stabilizes the helical conformation of the immature Gag lattice. This stabilization interferes with the access of the viral protease to its substrate cleavage site and prevents the formation of mature conical capsid cores. ☐ We have also investigated the interplay between cellular metabolite inositol hexakisphosphate (IP6) which promotes and drives assembly of immature HIV-1 Gag protein assemblies. We analyzed the structure of crystalline CACTD-SP1/IP6 in the presence and absence of BVM using MAS NMR. We obtained a large number of distance restraints and calculated the structure of CACTD-SP1/BVM/IP6 and CACTD-SP1/IP6 crystalline arrays to the precision backbone r.m.s.d. (root mean square deviation) of 0.7 Å. Simultaneous binding of BVM and IP6 and orientation of BVM inside the 6-helix bundle were discerned based on protein-ligand correlations observed in NMR spectra. We also analyzed complexes of BVM and CACTD-SP1 harboring amino-acid variants that confer resistance to BVM. Our results reveal a novel allosteric mechanism by which BVM blocks the pore and arrests viral maturation and will be important to guide the next generation of novel maturation inhibitors with increased potency. ☐ Chapter 3 describes the determination of the atomic resolution structure of the SARS-CoV-2 nucleocapsid (N) N-terminal domain (NTD) protein. The N protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in RNA packaging and hence, in viral infection, replication, and pathogenicity. NNTD facilitates the majority of RNA binding activity. Thus, NNTD represents a potential target for developing small-molecule inhibitors and a possible antigen epitope for vaccine development. We determined the atomic resolution structure of crystalline NNTD using high-frequency MAS NMR spectroscopy combined with X-ray diffraction. Our combined approach yields atomic resolution details of critical regions, previously inaccessible in NNTD structures, such as the intrinsically disordered residues at the N terminus and the functionally important β-hairpin loop. In addition, applying ultrafast (100 kHz) MAS permits assignment of side chain protons, not available by other means. The present structures provide important guidance for developing therapeutic interventions against SARS-CoV-2 infection. ☐ Chapters 4 and 5 describe implementation of DFT cluster calculations for accurate determination of NMR parameters in pharmaceutical compounds and an HIV-1 capsid protein. In chapter 4, the DFT calculation of 1H, 13C, and 15N chemical shifts is presented for Posaconazole, an antifungal drug. The calculation provided important information on intermolecular distances and helped the chemical shifts assignment of the MAS NMR spectra. In chapter 5, my contribution to the project is the implementation of DFT for accurate calculation of 19F NMR chemical shifts. Fluorosubstituted tryptophans whose crystal structures are known were used as benchmarks. Our results indicate that using a hybrid PBE0 functional with a 50% Hartree-Fock (HF) exchange term, chemical shift tensors can be calculated with reasonable accuracy. I incorporated this approach for 19F chemical shift calculations in HIV-1 capsid protein assemblies. A 6-Å spherical cluster around the central fluorine atom was found to yield moderate agreement between calculated and experimental chemical shift tensors of 19F atoms. Our results suggest that, to reach higher accuracy and precision in calculations of 19F chemical shift tensors in protein assemblies, larger cluster size and/or development of new DFT functionals would be required.
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Keywords
Biomolecular NMR, Density functional theory, HIV-1 maturation inhibitors, Nuclear magnetic resonance, SARS-CoV-2, Structural biology, COVID 19, Coronavirus disease 2019