Show Abstract ▼

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.

Open Access

Show Abstract ▼

Abstract: In this paper a practical solution for the reconstruction and segmentation of low-contrast X-ray tomographic data of protein crystals from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source is provided. The resulting segmented data will provide the path lengths through both diffracting and non-diffracting materials as basis for analytical absorption corrections for X-ray diffraction data taken in the same sample environment ahead of the tomography experiment. X-ray tomography data from protein crystals can be difficult to analyse due to very low or absent contrast between the different materials: the crystal, the sample holder and the surrounding mother liquor. The proposed data processing pipeline consists of two major sequential operations: model-based iterative reconstruction to improve contrast and minimize the influence of noise and artefacts, followed by segmentation. The segmentation aims to partition the reconstructed data into four phases: the crystal, mother liquor, loop and vacuum. In this study three different semi-automated segmentation methods are experimented with by using Gaussian mixture models, geodesic distance thresholding and a novel morphological method, RegionGrow, implemented specifically for the task. The complete reconstruction-segmentation pipeline is integrated into the MPI-based data analysis and reconstruction framework Savu, which is used to reduce computation time through parallelization across a computing cluster and makes the developed methods easily accessible.

Open Access

Show Abstract ▼

Abstract: Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 Å. To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Open Access

Show Abstract ▼

Abstract: The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi. We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.

Open Access

Show Abstract ▼

Abstract: K2P potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric K2Ps is unknown. Here, combining K2P2.1 (TREK-1) x-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and electrophysiology, we uncover unprecedented, asymmetric, potassium-dependent conformational changes that underlie K2P C-type gating. These asymmetric order-disorder transitions, enabled by the K2P heterodimeric architecture, encompass pinching and dilation, disrupt the S1 and S2 ion binding sites, require the uniquely long K2P SF2-M4 loop and conserved “M3 glutamate network,” and are suppressed by the K2P C-type gate activator ML335. These findings demonstrate that two distinct C-type gating mechanisms can operate in one channel and underscore the SF2-M4 loop as a target for K2P channel modulator development.