Expression, Purification, Characterization, and Solution Nuclear Magnetic Resonance Study of Highly Deuterated Yeast Cytochrome <i>c</i> Peroxidase with Enhanced Solubility

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome <i>c</i> peroxidase (CcP) constructs with enhanced solubility. Using a high-yield <i>Escherichia coli</i> expression system, we routinely produced uniformly labeled [²H,¹³C,¹⁵N]­CcP samples with high levels of deuterium incorporation (96–99%) and good yields (30–60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome <i>c</i>, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy

Probing the N‑Terminal β‑Sheet Conversion in the Crystal Structure of the Human Prion Protein Bound to a Nanobody

Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP^C, into its misfolded oligomeric form, known as prion or PrP^Sc. The canonical human PrP^C (HuPrP) fold features an unstructured N-terminal part (residues 23–124) and a well-defined C-terminal globular domain (residues 125–231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113–120) plays an important role in the conversion to PrP^Sc. The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel β-sheet with the β1 and β2 strands, demonstrating that the full-length HuPrP^C can adopt a more elaborate β0-β1-α1-β2-α2-α3 structural organization than the canonical β1-α1-β2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates β-enrichment in the PrP^C monomer as one of the early events in the conversion of PrP^C into PrP^Sc

Discovery, Structure–Activity Relationship, and Binding Mode of an Imidazo[1,2‑<i>a</i>]pyridine Series of Autotaxin Inhibitors

Autotaxin (ATX) is a secreted enzyme playing a major role in the production of lysophosphatidic acid (LPA) in blood through hydrolysis of lysophosphatidyl choline (LPC). The ATX–LPA signaling axis arouses a high interest in the drug discovery industry as it has been implicated in several diseases including cancer, fibrotic diseases, and inflammation, among others. An imidazo­[1,2-<i>a</i>]­pyridine series of ATX inhibitors was identified out of a high-throughput screening (HTS). A cocrystal structure with one of these compounds and ATX revealed a novel binding mode with occupancy of the hydrophobic pocket and channel of ATX but no interaction with zinc ions of the catalytic site. Exploration of the structure–activity relationship led to compounds displaying high activity in biochemical and plasma assays, exemplified by compound <b>40</b>. Compound <b>40</b> was also able to decrease the plasma LPA levels upon oral administration to rats