Browsing by Subject "X-ray crystallography"

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    Fragment based inhibitor design of Mycobacterium tuberculosis BioA
    (2015-01) Dai, Ran
    7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis (Mtb) is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). We herein report our fragment based inhibitor design of Mtb BioA. Using differential scanning fluorimetry (DSF) fragment screening, the Maybridge Ro3 library of 1000 molecules was screened. Twenty-one compounds giving rise to Tm shifts exceeding ±2°C were then investigated in crystallographic experiments. Six fragments have been co-crystallized with BioA to characterize binding. Each compound has a unique binding mode, and subtle variations in ligand binding site geometry are induced upon binding of different fragment molecules. Binding affinities of the fragments were characterized via isothermal titration calorimetry (ITC). A fragment extension strategy was used to rationally optimize these fragment hits. A commerce based SAR was used and identified 50 compounds containing the core of one of the fragments. These compounds were further screened virtually and experimently by DSF. Four optimized BioA ligands from fragment optimization were validated by X-ray crystallography, including a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5′-phosphate (PLP) cofactor. Binding affinities of these ligands have been characterized by ITC or kinetic assay. The six fragment complex structures were also used for optimization of HTS lead compounds. Six HTS lead compounds were co-crystallized with BioA at high resolution. Design of optimized compounds was by overlapping the fragments and HTS lead binding conformations in the BioA active site. Molecules predicted to have better potency were proposed. Two N-aryl piperazine inhibitors of BioA from HTS optimization were characterized using X-ray crystallography and ITC. One inhibitor that combines features of one HTS lead and one fragment was confirmed with improved binding affinity by ITC.
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    Ion-sensing properties of ferrocenyl-diaminomaleonitrile derivatives.
    (2010-07) Dahlby, Michael Raymond
    Mono- (1) and bis (2) -substituted Shiff bases obtained by the reaction between ferrocenecarbaldehyde and diaminomaleonitrile were prepared and characterized by 1H and 13C NMR, IR, UV-vis spectroscopy methods, elemental analysis, and X-ray crystallography. Electrochemical techniques were employed to investigate the possibility of a mixed-valence state of the iron centers in 2, which are connected by a conjugated π-system. Two reversible oxidation potentials were observed in cyclic voltammetry, and spectroelectrochemical experiments revealed the presence of low-energy inter-valence charge transfer band in 2+, corresponding to a weakly-coupled class II system. Both complexes were tested for cation-recognition properties using a variety of main group and transition-metal cations. It was shown that the complex 1 can be used as a selective optical sensor in recognition of Hg(II) and Ag(I) ions.
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    Mechanistic and structural studies of the non-heme iron oxygenase enzymes protocatechuate 3,4-dioxygenase, CmlA, and CmlI
    (2015-12) Knoot, Cory
    Oxygenase enzymes catalyze a remarkable variety of challenging biological transformations using molecular dioxygen (O2) as a cosubstrate. Oxygenases make possible the direct reaction of ground-state triplet O2 with organic singlet substrates, a transformation that is spin-forbidden by quantum mechanics. The set of chemical processes by which enzymes overcome this forbidden reaction are collectively termed ‘O2 activation.’ The result of oxygenase reactions is incorporation of one or both atoms of O from O2 into the organic substrate. Because of the critical role of oxygenases in many fundamental biological processes, an understanding of the catalytic and regulatory mechanisms that underlie O2 activation is required for the development of new medical, industrial, ecological and agricultural technologies. Non-heme iron oxygenases use mononuclear iron or diiron cofactors that are not coordinated in a porphyrin scaffold. This dissertation focuses on structural and mechanistic studies of three non-heme iron enzymes. The first, protocatechuate 3,4-dioxygenase (3,4-PCD), is an archetypal aromatic ring-cleaving oxygenase from soil bacteria that uses the oxidized form (Fe3+) of the iron cofactor to react with O2. It is a member of one of only two enzyme families that use Fe3+ to activate O2. CmlA is a diiron cluster-containing oxygenase that is involved in the non-ribosomal peptide synthetase-mediated biosynthesis of the antibiotic chloramphenicol. It catalyzes the first step in this pathway: the β-hydroxylation of an amino acid precursor of the antibiotic. The third enzyme, CmlI, also uses a diiron cluster cofactor and catalyzes the final step in chloramphenicol biosynthesis by converting the arylamine group of the chloramphenicol precursor to its arylnitro analog. Herein, we report the determination of the X-ray crystal structures of the two most critical oxygenated intermediates in the 3,4-PCD catalytic cycle and characterize the reaction of the enzyme with diagnostic substrates. This work confirms mechanistic proposals that have existed since the 1960s and lays the groundwork for future studies. In the last two chapters of the dissertation, we report the X-ray crystal structures of CmlA and CmlI and compare their structures to other diiron cluster-utilizing enzymes. We also discuss the insights gained into their catalytic and regulatory mechanisms.
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    Receptor recognition of SARS-CoV-2 and vaccine design
    (2022-07) Geng, Qibin
    SARS-CoV-2, the causative agent of COVID-19, has devastated the world with many waves of variants of concern (VOC) over these two years. The current highly contagious and fast-spreading omicron variants of SARS-CoV-2 infect the respiratory tracts efficiently. The receptor-binding domain (RBD) of the omicron spike protein recognizes human ACE2 as its receptor and plays a critical role in the tissue tropism of SARS-CoV-2. Here, we showed that the omicron RBD binds to ACE2 more strongly than does the prototypic RBD from the original Wuhan strain. We also measured how individual omicron mutations affect ACE2 binding. We further determined the crystal structure of the RBD complexed with ACE2 at 2.6 Å. The structure shows that omicron mutations caused significant structural rearrangement of two mutational hotspots at the RBD/ACE2 interface, elucidating how each omicron mutation affects ACE2 binding. The enhanced ACE2 binding by the omicron RBD can facilitate the omicron variant to infect the respiratory tracts where the ACE2 expression level is low. Our study provides insights into the receptor recognition and tissue tropism of the omicron variant. Besides the SARS-CoV-2 omicron variant, other VOCs, such as alpha, beta, gamma, and delta variants, are also associated with increased transmissibility and virulence, and the possible emergence of escape mutations. Studies have indicated that intermediate horseshoe bats and pangolins are potential reservoirs of SARS-CoV-2. Furthermore, recent evidence also shows that SARS-CoV-2 is able to jump between humans and minks, which raises concerns about the prevention and control of SARS-CoV-2, as the spillover of SARS-CoV-2 is associated with potential mutations. Here we measure the binding affinities of SARS-CoV-2 VOC RBDs to human mink, pangolin, or intermediate horseshoe bat ACE2s by surface plasmon resonance (SPR) assay, and find that SARS-CoV-2 VOC RBDs can bind to mink, pangolin, and intermediate horseshoe bat ACE2s with a differential level of affinities, albeit human ACE2 has the highest binding affinities. To elucidate the detailed interaction mechanisms, besides the crystal structure of the Omicron (BA.1) RBD with human ACE2, the crystal structures of the complexes of SARS-CoV-2 VOC-specific RBD with human ACE2, and SARS-CoV-2 RBD with pangolin and mink ACE2s are determined at 2.61 Å, 2.98 Å, and 2.76 Å, respectively. These structures adopt similar binding patterns with two hotspot bind sites in the RBD/ACE2 interface. The residue variations of the two hotspots in the RBD/ACE2 interface are mainly responsible for the differential binding affinities of SARS-CoV-2 variant RBDs to human ACE2, and SARS-CoV-2 RBD to pangolin and mink ACE2s. We further measure the binding affinities and provide structural analysis of the species-specific SARS-CoV-2 RBD mutations to the binding of cross-species ACE2s by SPR. Overall, this study provides more structural evidence of the interaction of SARS-CoV-2 variant RBDs with human ACE2, and cross-recognition of SARS-CoV-2 RBD to pangolin and mink ACE2s. The facts that SARS-CoV-2 VOCs can bind to other species’ ACE2s, and species-specific SARS-CoV-2 RBD mutations differentially impact cross-species ACE2s’ binding affinities, highlight the need for surveillance of the viral genome from infected animals and humans, particularly the genome regions affecting diagnostic tests, antiviral therapies, and vaccine development.The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS- CoV-2 from attaching to its host receptor and potently neutralized the cell entry of various SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during a two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from the SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2. In summary, the dissertation provides the structural basis for the receptor recognition of the SARS-CoV-2 variants of concern RBDs to human ACE2, and cross-species recognition of SARS-CoV-2 RBD to pangolin and mink ACE2s. The binding affinity determination and analysis of SARS-CoV-2 VOC RBDs and species-specific SARS-CoV-2 RBD mutations to cross-species ACE2s will help to elucidate the detailed interaction mechanism. Finally, the dissertation proposes and develops an effective novel virus-like particle vaccine against COVID-19.
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    Structural enzymology of soluble methane monooxygenase protein-protein interactions.
    (2021-05) Jones, Jason
    Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that activates molecular oxygen, breaks the 105 kcal/mol C-H bond of CH4, and inserts one atom of O to create methanol at ambient temperature and pressure. This feat of catalytic prowess requires all three protein components of sMMO for efficient multiple turnover catalysis: the hydroxylase (sMMOH), the reductase (MMOR), and the regulatory protein (MMOB). The structural mechanism of how these sMMO components interact to regulate the formation and decay of the chemical intermediates of the reaction cycle is not well understood. Our recent advances in sMMOH expression and purification have allowed us to obtain protein crystals of the sMMOH:MMOB complex. Using X-rays generated by either an X-ray free electron laser at room temperature or a synchrotron at 100 K, we obtained high resolution structures of the Methylosinus trichosporium OB3b sMMOH:MMOB complex for the first time. Analysis of the data shows in great detail how MMOB modulates the structure of sMMOH during the steps leading up to O2 binding. New insight is gained about the path O2 and methane take into the sMMOH active site, and how the selectivity and timing of this entry is controlled by MMOB in the sMMOH:MMOB complex. Additionally, biosynthetic incorporation of 5-fluorotryptophan into MMOB and MMOR, as well as post-translational modification of an MMOB variant with a trifluoroacetone probe, allowed us to use 1D-19F-NMR to investigate the complex series of sMMO protein interactions that regulate the beginning of the sMMO catalytic cycle. A new model emerges describing how sMMO protein component affinities and exchange from protein-protein complexes control the dynamics of reaction cycle intermediates to drive catalysis.