| 1. |
- John, Juliane, 1987-
(författare)
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High (valent) on O2 : Ribonucleotide Reductase and Methane Monooxygenase
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Macromolecular X-ray crystallography (MX) is a powerful method to investigate protein structures. However, proteins with redox-active centres and radicals are very susceptible to photoreduction. It is therefore challenging to acquire structural details of redox-active centres in defined oxidation states or protein radicals using synchrotron radiation. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) radiation mitigates this problem. XFELs produce intense pulses of femtosecond length that give rise to diffraction before photoinduced movement can occur in the illuminated protein. Additionally, SFX allows experiments at room temperature and induction of reactions in crystallo. In this thesis two different redox-active enzyme systems were investigated with MX and SFX. The first part examines ribonucleotide reductase (RNR). RNR is the only known enzyme to synthesize de novo deoxyribonucleotides, the building blocks of DNA. Class I RNR consists of a small subunit R2 and a large subunit R1. R2 generates a radical in an oxygen dependent way and delivers it to R1 for ribonucleotide reduction. After catalysis the radical is transferred back to R2 until further use. Class I RNR is divided in five subclasses, mostly based on their mechanism of radical generation. In Paper I class Ib R2 is investigated. R2b binds two manganese ions that react with superoxide to produce a radical. The superoxide is provided by a small flavoprotein, NrdI, bound to R2. When exposed to molecular oxygen, reduced NrdI generates superoxide that is transferred to the R2 active site. Here two SFX structures of reduced and oxidized NrdI in complex with R2 are presented and it is suggested how the binding and NrdI oxidation state could influence the superoxide production. In Paper II the SFX structure of a R2e protein radical is presented. Class Ie R2 contains a metal-free active site. The comparison of the radical structure with a ground state structure highlights the changes induced by radical formation. A mechanism for the initiation of the radical transfer to R1 is proposed based on the structural details observed. In Paper III light is shed on a new variant of R2e. Three of the typically conserved active site residues are mutated in R2e; from three glutamates to valine, proline and lysine (VPK) or to glutamine, serine and lysine (QSK). Other publications, including Paper II, describe the VPK mutation but as of now the QSK variant has not been examined. Here, crystal structures of a R2e QSK protein are shown. A tyrosine close to the active site is post-translationally modified to a dihydroxyphenylalanine (DOPA). The amount of modified protein is shown to scale with the coexpression of other proteins of the RNR operon. The second redox-active enzyme investigated is soluble methane monooxygenase (sMMO). sMMO oxidizes methane to methanol and is produced by methanotrophs; bacteria that use methane as their sole carbon source. Methane is a potent greenhouse gas and can be found in ever increasing concentrations in the atmosphere due to human activities; sMMO is thus a compelling target for biotechnological development. Paper IV presents SFX structures of the catalytic subunit MMOH in complex with its small regulatory subunit MMOB in the oxidized and reduced resting state. It is also demonstrated that the complex can undergo the catalytic cycle in crystallo, allowing investigation of reaction cycle intermediates in the future.
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| 2. |
- Persson Košenina, Sara, 1993-
(författare)
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Botulinum neurotoxins
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Botulinum neurotoxins (BoNTs) are the most potent toxins known to man, with less than 1 μg of pure toxin being enough to kill an adult man. Despite the high toxicity, BoNTs are widely used in cosmetics and in medicine for the treatment of an increasing number of medical conditions.BoNTs have a conserved structure that consists of three domains (a receptor binding, translocation, and catalytic domain). The receptor binding domain is responsible for binding to neuronal receptors, the translocation domain is a delivery vehicle that transports the catalytic domain into the cytosol, where the latter cleaves its target - proteins of the SNARE family, inhibiting neurotransmitter release and consequently causing muscle paralysis.BoNTs are produced by the bacteria Clostridium botulinum together with several other accessory proteins, which are responsible for shielding BoNTs in the harsh environment of the target gastrointestinal tract and assisting them in crossing the epithelial barrier between the gastrointestinal tract and general circulation.Several BoNT serotypes (A-G) have been identified over the years. Additionally, several BoNT-like toxins have been identified in non-Clostridial types of bacteria. Namely, these proteins are BoNT/Wo, BoNT/En and PMP1.In this thesis, we present six papers, where we studied both the canonical BoNTs and the new BoNT-like toxins as well as their accessory proteins using structural biology techniques, such as X-ray crystallography and cryo-EM. Elucidating the structures of these proteins is crucial for understanding their function and mechanism of action.
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| 3. |
- Rehling, Daniel, 1985-
(författare)
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Structural Studies of Proteins involved in Nucleotide Metabolism : Studies of a ribonucleotide reductase from A. aeolicus and NUDT15
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is separated into two parts. The first part concerns ribonucleotide reductase from Aquifex aeolicus. A. aeolicus is a hyperthermophilic bacterium that thrives at extremely high temperatures of 80-95 °C. We present the X-ray crystal structures of both the R1 and R2 subunits of this protein, which represents the first structure of a class Ia ribonucleotide reductase from a hyperthermophile and the first structure of an R1 from the NrdAh phylogenetic subclass. Several novel features were seen in the R1 structure such as the simultaneous binding of two ATP molecules in the ATP-cone domain as well as a novel “β-hairpin hook” feature which spans the dimer interface of the R1 protein. The gene encoding the R2 protein contains a self-cleaving intein domain. We examined two constructs of this protein, one with the sequence of the intein removed at the DNA level and the wild-type construct. Both crystal structures were found to be identical, showing the efficient cleavage of the intein domain in the wild-type construct. The second part of this thesis concerns the NUDIX hydrolase NUDT15. The physiological function of NUDT15 is still unknown, however certain mutations in this gene are associated with thiopurine intolerance in patients. Thiopurines are chemotherapeutic drugs used in the treatment of diseases such as acute lymphoblastic leukemia, the most common type of childhood leukemia, and inflammatory bowel disease. Thiopurine drugs are converted by the cell to the active metabolite 6-thio-dGTP which can then act as a substrate for DNA polymerase. Incorporation of these anti-metabolites into DNA produces the desired cytotoxic effects. We show that NUDT15 breaks down the active metabolites of these drugs which leads to a lowered effective dose. The absence of a functioning NUDT15 protein in patients that have inactivating mutations in the gene coding for NUDT15 results in a drastically increased effective dose of these compounds. A normal dose of a thiopurine drug can lead to severe and possibly life-threatening complications in these patients. The role of NUDT15 in thiopurine metabolism is established by in vitro and cellular data as well as the X-ray crystal structure of NUDT15 in complex with 6-thio-GMP. Acyclovir and ganciclovir are two antiviral drugs whose mechanism of action is similar to that of thiopurines. These drugs are also metabolized to their tri-phosphorylated forms and are then preferentially incorporated into viral DNA. Here again, we use in vitro, cellular and structural data to show that NUDT15 breaks down the active metabolites of these drugs. Two separate and structurally distinct lines of potent inhibitors for NUDT15 were developed with support of crystallographic studies. We show that cells are sensitized to both thiopurine and antiviral treatments in the presence of these inhibitors. Binding of our inhibitors to NUDT15 provided substantial thermal stabilization. The stabilizing effect of inhibitor binding enabled us to solve structures of the four most clinically relevant NUDT15 variants, thus elucidating the structural basis for the thiopurine sensitivity phenotype.
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| 4. |
- Henriksson, Linda, 1973-
(författare)
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Structural and functional studies of a novel Botulinum neurotoxin and of MTH1
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- X-ray crystallography visualizes the three dimensional molecular structures of proteins at atomic resolution. Seeing the molecular structure of a biomedically interesting protein enables a higher understanding of its function. The process of producing pure protein from genetic material to generate crystals and determine the molecular structure can be a long and challenging process. My thesis involves structural and functional studies of two different proteins, which are both biomedically interesting and important to learn about. X-ray crystallography is the method which has been used to determine the majority of the protein structures that we know of today and is also the method used in the results presented in my thesis. Today there are no cancer therapies defeating all types of cancers and they do not come without side effects. Battling cancer diseases often include long and painful treatments. Finding an anti-cancer drug targeting phenotypes characteristic of cancer cells is a compelling thought. MutT homolog-1 (MTH1) is an enzyme present in all proliferating cells. The enzyme seems to be crucial for cancer cell survival but not for the viability of normal cells. MTH1 cleans out oxidized and thereby damaged nucleotides from the free nucleotide pool and stops them from being used in DNA synthesis. This process is very important in fast proliferating cancer cells. The hypothesis is to inhibit MTH1 and thereby allow a limitless amount of DNA damage in the cancer cells. This action will eventually kill cancer cells while not affecting normal cells. The molecular structure of MTH1 with (PDB ID: 3ZR0) and without a product bound (PDB ID: 3ZR1) was determined and is presented in my thesis. These two structures aided in the synthesis of inhibitors. Botulinum neurotoxins (BoNTs) are the most potent toxins known. As little as one gram of pure toxin could potentially kill one million people. Due to its potency BoNT is a potential bioterrorism threat. The toxin is also a very potent drug used clinically to relieve the symptoms of an array of neuromuscular disorders. Most people know this neurotoxin by one of its commercial names: Botox™. Additionally BoNTs are the cause of botulism. BoNTs are neuro-specific enzymes that target neuromuscular signaling, inducing flaccid paralysis and potentially death. It is of importance to learn more about these toxins to enable the development of new countermeasures, vaccines or more efficient neuroparalytic drugs. BoNTs consist of three domains with different functions, all crucial for intoxication. The toxins are fragile and can easily be destroyed by harsh surroundings if not protected by non-toxic non-hemagglutinin (NTNH) proteins. The complex of some BoNT serotypes and their protective NTNH have proven to be pH-dependent. Parts of the intoxication process are not yet clear and their mechanisms are still puzzling researchers. Until recently seven BoNT serotypes were identified. We have now identified and characterized a novel serotype called BoNT/X. The molecular structure of the active domain is presented here (PDB ID: 6F47). The pH-dependent mechanism forming a complex as seen in other serotypes, is confirmed to be present in BoNT/X as well.
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| 5. |
- Massad, Tariq, 1979-
(författare)
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Structural Studies of Flexible Biomolecules and a DNA-binding Protein
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.
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