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Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. CCHFV is shortlisted as a priority pathogen by the World Health Organization. It is a (-)ssRNA virus of the family Nairoviridae in the Bunyavirales order. The CCHFV membrane fusion glycoprotein Gc has been shown to be the main target of the host neutralizing antibody response, whereas a secreted glycoprotein GP38 was demonstrated to be the target of non-neutralizing protective antibodies. However, structural information on the viral glycoproteins as well as antibody-mediated neutralization mechanisms has been lacking for members of the nairovirus family. The first part of this thesis describes the structure of monomeric prefusion CCHFV Gc bound to the antigen binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show that the two Fabs act in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The above structures also revealed the neutralization mechanism of antibodies targeting 6 different antigenic sites on CCHFV Gc, a few of which were further validated with structural studies. Describing CCHFV neutralization at the mechanistic level, our data guide the design of future therapeutic antibodies and will likewise support the design of protective CCHFV vaccines. The second part of this thesis presents the structure of GP38, which reveals a novel protein fold. This study also describes the structure of a mouse antibody 13G8 bound to GP38; this antibody has been shown to protect against CCHFV. Mapping of the epitopes of several human GP38 antibodies onto the GP38 structure shows competition with the 13G8 epitope and identification of additional antigenic sites. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. Collectively both these studies provide the molecular underpinnings essential for developing CCHFV-specific medical countermeasures for epidemic preparedness.

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Abstract: Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.

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Abstract: Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an "undruggable" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors.

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Abstract: The flow of potassium ions across the cell membrane is regulated by K2P potassium channels, controlling electrical activity in the brain, heart and nervous system. These channels have important roles in pain, migraine, depression, anaesthetic responses, arrhythmias, hypertension and lung injury responses. K channels regulate ion flow using a selectivity filter (C-type) gate. However, previous 2P structural studies have not captured the gating mechanisms, and they remain poorly understood. Currently, there are no drugs available that selectively target K2P potassium channels. To develop new ways to control their function, we need to understand more about the gating mechanisms and their roles in human biology. In work published in Science Advances, researchers from Diamond Light Source, University of California, San Francisco and the University Pittsburgh, USA, combined X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics and electrophysiology to uncover the changes that occur in K2P C-type gating. At Diamond, the team used the Long-Wavelength Macromolecular Crystallography (MX) beamline (I23) to observe changes in potassium ions within the channel directly. I23 is the only beamline in the world optimised to measure signals from potassium ions directly, and its unique capabilities were central to these studies. The results show that K2P gating involves pinching and dilation of two key elements of the channel selectivity filter gate. These structural changes are accompanied by the loss of potassium ions. Including a small molecule activator (ML335) suppressed these changes and demonstrated how stabilisation of the selectivity filter gate facilitates ion flow through the channel. These studies show that small molecule activators bind to and stabilise the K2P selectivity filter gate, preventing the pinching and dilation conformational changes and loss of potassium ions that lead to channel inactivation. These findings open a path to develop new K2P channel- directed drugs to treat pain, ischemia (restricted blood flow), depression and lung injury.

Open Access

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Abstract: During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.