| 1. |
- Gustafsson, Robert, 1988-
(författare)
-
Structural and functional studies of proteins of medical relevance : Protein-ligand complexes in cancer and novel structural folds in bacteria
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- X-ray crystallography is a tool for determining the structures of proteins and protein-ligand complexes. In this thesis the method has been employed to study several proteins of medical relevance.Cancer is a terrible disease, severely impacting those affected, as well as their family and friends. Current cancer treatments involve a combination of cytostatic drugs, surgery and radiation treatment. Unfortunately many cytostatic drugs also kill healthy cells, which gives rise to serious side-effects. The discovery of treatments which selectively inhibit proteins essential for cancer cell survival but which are non-essential in normal cells, could reduce such side-effects.MTH1 is a protein that degrades oxidised nucleotides, which when incorporated into DNA cause mutations and subsequent cell death. Cancer cells have higher levels of reactive oxygen species, which create oxidised nucleotides. In Paper I it was discovered that cancer cells are dependent on MTH1 for their survival. Crystal structures of MTH1 in complex with small molecules guided their development into potent MTH1 inhibitors, capable of killing cancer cells. Cells with increased amounts of oxidised nucleotides, or with induced hypoxia, were more susceptible to MTH1 inhibition, as shown in Paper II. In Paper III several MTH1 orthologues from organisms often used in pre-clinical studies were tested for MTH1 inhibition. Leucine 116 of mouse MTH1 was determined to be important for the lower inhibition of the developed inhibitors towards this enzyme. A virtual fragment screening study using commercial chemicals resulted in several potent MTH1 inhibitors, as shown in Paper IV. The crystal structures with the fragments or optimised inhibitors did in most cases agree with the docking pose determined from the virtual screening. In addition to the known function of MTH1 in the degradation of oxidised nucleotides, Paper V showed that MTH1 also degrades methylated nucleotides.MTHFD2 is responsible for providing one-carbon units for nucleotide synthesis in cancer cells. As MTHFD2 is present in cancer cells but not in healthy cells, targeting the enzyme would make it possible to selectively kill cancer cells. Paper VI presents the first structure of MTHFD2, along with the first inhibitor of the protein. This information provides a starting point for the development of potent and selective MTHFD2 inhibitors.The botulinum neurotoxin from the bacterium Clostridium Botulinum is the causative agent of the deadly disease botulism. The action of the botulinum neurotoxin on nerve cells results in paralysis, and is life-threatening if the patient is not helped with breathing support. However, low doses of the neurotoxin are used as a successful treatment for several medical conditions, such as involuntary spasms. In Paper VII the structure of two proteins, P47 and OrfX2, encoded in the gene cluster of a botulinum neurotoxin, were determined. The structures resembled tubular lipid-binding proteins, previously only found in eukaryotes. The proteins were also found to be able to bind lipids. This work gives new insight into the structure and function of this group of proteins, which help the deadly botulinum neurotoxins.
|
|
| 2. |
- Henriksson, Linda, 1973-
(författare)
-
Structural and functional studies of a novel Botulinum neurotoxin and of MTH1
- 2018
-
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.
|
|
| 3. |
- Martínez-Carranza, Markel, 1992-
(författare)
-
Structural Insights into Botulinum Neurotoxins and the ALFA-tag System : Structural and Functional Studies of Proteins Related to the Botulinum Neurotoxins and Design of a Novel Epitope Tag
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is divided into two sections; the first part describes our work in the field of botulinum neurotoxins (presented in papers I, II, III, and manuscript IV) and the second part summarizes our work involving the design of a new biochemical tool (presented in paper V).Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to date. They have a conserved structure that consists of three domains (receptor-binding, translocation, and catalytic domain), each of which has a distinct function. The receptor-binding domain binds to neuronal receptors, and after endocytosis the translocation domain shuttles the catalytic domain into the cytosol, where it cleaves neuronal proteins of the SNARE family, which are part of the vesicle-membrane fusion machinery.In paper I, we studied proteins of unknown function (OrfX1, OrfX2, OrfX3, and P47), which are co-expressed with certain BoNTs. We solved the crystal structures of OrfX2 and P47, and their structural resemblance to tubular lipid binding proteins (TULIP) together with lipid binding studies, led us to conclude that OrfX1 and P47 are able to bind phosphatidyl inositol phosphates (PIPs) in vitro.In paper II, we studied the binding of BoNT/B, /DC and /G to their protein receptor synaptotagmin (Syt). We determined their affinities to synaptotagmins from different species, and concluded that residue F50 in bovine Syt-II is responsible for its increased affinity towards BoNT/DC. In addition, we studied the interaction between BoNT/G and Syt-II via STD-NMR. Our results showed the binding to be similar to BoNT/B and Syt-II, and that the N-terminal region of the Syt peptide is important for the binding of BoNTs to synaptotagmin, even though it is not part of the binding interface.In paper III and manuscript IV, we present the identification of a novel BoNT serotype named BoNT/X. We showed that BoNT/X cleaves the non-canonical substrates VAMP4, VAMP5 and Ykt6, as well as the canonical substrate VAMP1-3 at a new cleavage site, distinct from other BoNTs. In addition, we present the cryo-EM structure of BoNT/X in complex with its non-toxic interaction partner NTNH. Our pH stability experiments revealed that BoNT/X-NTNH remain bound at neutral to moderately high pH, in contrast with what is observed for BoNT/A-NTNH.In paper V we present the design of a novel epitope tag named the ALFA system. The ALFA tag is a short α-helical protein tag that is highly stable and electroneutral. The ALFA nanobody has a very high affinity for the tag and is small enough to allow for high performance in high-resolution microscopy. The crystal structure of the ALFA nanobody in complex with the tag led to a modified version of the ALFA nanobody that can release the tag via competitive elution with free ALFA peptide. Our results showed that this system outperforms several commercially available systems in protein purification and high-resolution microscopy.
|
|
| 4. |
- Ravishankar, Harsha
(författare)
-
Characterization of membrane protein active transport under native-like conditions
- 2018
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- P-type ATPase proteins are a family of membrane proteins that maintain concentration gradients of e.g. ions by ATP-driven transport across the membrane. While these transporters share many features in their molecular architecture, structural differences are required to convey ion specificity. In addition, the transport dynamics accomplished by conformational changes may also differ in-between ATPase subtypes. Therefore, resolving P-type ATPase temporal and spatial structural dynamics is crucial to understand how these proteins function. To pave way for time-resolved X-ray characterization of conformational changes during P-type ATPase transport in solution, it was necessary to identify optimal conditions for triggering the protein reaction. Therefore, ATP activation of a recombinant Zn2+-transporting ATPase was studied using a biochemical activity assay and infrared spectroscopic techniques. Specifically, time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy was used to study activation using photolysis of caged ATP. The highest protein activity was obtained at a protein concentration of 25 mg/mL at 310 K and pH 7, and this required the presence of 20% glycerol as a stabilizing agent. It was also observed that neither the presence of caged ATP nor higher lipid concentrations affected protein activity significantly. The Ca2+-transporting sarcoplasmic reticulum ATPase (SERCA), found abundantly in skeletal muscle native membranes, was used to develop the time-resolved Wide-Angle X-ray Scattering (TR-WAXS) technique for irreversible caged ATP activation and subsequent structural refinement. Several SERCA intermediate states and protein-lipid interactions have been characterized by X-ray crystallography, rendering the SERCA protein an ideal proof-of-principle target system. In the native membrane, fast single-cycle dynamics were registered followed by steady state accumulation. The structural refinement procedure starting from existing intermediate crystal structures indicated that the accumulated state represented a phosphorylated state (E2-P) or possibly a Ca2+ bound E2 state (Ca2E2P), which has so far eluded X-ray crystallographic characterization. The results also showed that the corresponding ground state (Ca2E1) underwent significant rearrangements of the cytosolic domains, which implies that the Ca2E1 crystal structure might be one of several possible structures and might not represent the dominant structure in solution. Additionally, the TR-WAXS models indicated that the rocking motion of the soluble domains observed in a detergent/lipid mixture is also present in the native membrane.
|
|
