Biochemistry and Pharmacology - Research Publications

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    Reaction mechanism of the bioluminescent protein mnemiopsin1 revealed by X-ray crystallography and QM/MM simulations
    Molakarimi, M ; Gorman, MA ; Mohseni, A ; Pashandi, Z ; Taghdir, M ; Naderi-Manesh, H ; Sajedi, RH ; Parker, MW (ELSEVIER, 2019-01-04)
    Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2+-dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd2+-loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156-Arg-39-Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramide-binding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family.
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    A structural view of PA2G4 isoforms with opposing functions in cancer
    Stevenson, BW ; Gorman, MA ; Koach, J ; Cheung, BB ; Marshall, GM ; Parker, MW ; Holien, JK (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2020-11-20)
    The role of proliferation-associated protein 2G4 (PA2G4), alternatively known as ErbB3-binding protein 1 (EBP1), in cancer has become apparent over the past 20 years. PA2G4 expression levels are correlated with prognosis in a range of human cancers, including neuroblastoma, cervical, brain, breast, prostate, pancreatic, hepatocellular, and other tumors. There are two PA2G4 isoforms, PA2G4-p42 and PA2G4-p48, and although both isoforms of PA2G4 regulate cellular growth and differentiation, these isoforms often have opposing roles depending on the context. Therefore, PA2G4 can function either as a contextual tumor suppressor or as an oncogene, depending on the tissue being studied. However, it is unclear how distinct structural features of the two PA2G4 isoforms translate into different functional outcomes. In this review, we examine published structures to identify important structural and functional components of PA2G4 and consider how they may explain its crucial role in the malignant phenotype. We will highlight the lysine-rich regions, protein-protein interaction sites, and post-translational modifications of the two PA2G4 isoforms and relate these to the functional cellular role of PA2G4. These data will enable a better understanding of the function and structure relationship of the two PA2G4 isoforms and highlight the care that will need to be undertaken for those who wish to conduct isoform-specific structure-based drug design campaigns.
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    From Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae
    Dogovski, C ; Gorman, MA ; Ketaren, NE ; Praszkier, J ; Zammit, LM ; Mertens, HD ; Bryant, G ; Yang, J ; Griffin, MDW ; Pearce, FG ; Gerrard, JA ; Jameson, GB ; Parker, MW ; Robins-Browne, RM ; Perugini, MA ; Taylor, P (PUBLIC LIBRARY SCIENCE, 2013-12-13)
    Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.
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    The Structural Basis for a Transition State That Regulates Pore Formation in a Bacterial Toxin
    Wade, KR ; Lawrence, SL ; Farrand, AJ ; Hotze, EM ; Kuiper, MJ ; Gorman, MA ; Christie, MP ; Panjikar, S ; Morton, CJ ; Parker, MW ; Tweten, RK ; Johnson, EA (AMER SOC MICROBIOLOGY, 2019-03-01)
    The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning β-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other β-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning β-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large β-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.
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    Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding
    Langendorf, CG ; Ngoei, KRW ; Scott, JW ; Ling, NXY ; Issa, SMA ; Gorman, MA ; Parker, MW ; Sakamoto, K ; Oakhill, JS ; Kemp, BE (NATURE PUBLISHING GROUP, 2016-03-01)
    The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.
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    QM/MM simulations provide insight into the mechanism of bioluminescence triggering in ctenophore photoproteins
    Molakarimi, M ; Mohseni, A ; Taghdir, M ; Pashandi, Z ; Gorman, MA ; Parker, MW ; Naderi-Manesh, H ; Sajedi, RH ; Khodarahmi, R (PUBLIC LIBRARY SCIENCE, 2017-08-04)
    Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C3 position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent.
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    Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation
    Lawrence, SL ; Feil, SC ; Morton, CJ ; Farrand, AJ ; Mulhern, TD ; Gorman, MA ; Wade, KR ; Tweten, RK ; Parker, MW (NATURE PUBLISHING GROUP, 2015-09-25)
    Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.