Open Access

Show Abstract ▼

Abstract: Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK – to a much larger extent than Trypanosoma PFK – is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10‐fold, compared to only a 1.3‐ and 1.4‐fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme’s ‘reaching arms’ and ‘embracing arms’ that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T‐to‐R equilibria for the two families, with the low‐melting Leishmania PFK favouring the flexible inactive T‐state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP‐dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.

Show Abstract ▼

Abstract: Heterocycles, a class of molecules that includes oxazoles, constitute one of the most common building blocks in current pharmaceuticals and are common in medicinally important natural products. The antitumor natural product nataxazole is a model for a large class of benzoxazole containing molecules that are made by a pathway that is not characterized. We report structural, biochemical and chemical evidence that benzoxazole biosynthesis proceeds through an ester generated by an ATP dependent adenylating enzyme. The ester re‐arranges via a tetrahedral hemiorthoamide to yield an amide which is a shunt product and not, as previously thought, an intermediate in the pathway. A second zinc dependent enzyme catalyses formation of hemiorthoamide from the ester but, by shuttling protons, the enzyme eliminates water, a reverse hydrolysis reaction, to yield the benzoxazole and avoids the amide. These insights have allowed us to harness the pathway to synthesize a series of novel halogenated benzoxazoles.

Show Abstract ▼

Abstract: The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin, came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high resolution structures (≤ 1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid; and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µM), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which up-regulation of Wnt signalling may be beneficial.

Open Access

Show Abstract ▼

Abstract: Protein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.

Open Access

Show Abstract ▼

Abstract: Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the β-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin β-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.