Trichomonas vaginalis , the causative agent of trichomoniasis, is a prevalent anaerobic protozoan parasite responsible for the most common nonviral sexually transmitted infection globally. While ...metronidazole and its derivatives are approved drugs for this infection, rising resistance necessitates the exploration of new antiparasitic therapies. Protein posttranslational modifications (PTMs) play crucial roles in cellular processes, and among them, hypusination, involving eukaryotic translation factor 5A (eIF5A), has profound implications. Despite extensive studies in various organisms, the role of hypusination in T. vaginalis and its potential impact on parasite biology and pathogenicity remain poorly understood. This study aims to unravel the structural basis of the hypusination pathway in T. vaginalis using X‐ray crystallography and cryo‐electron microscopy. The results reveal high structural homology between T. vaginalis and human orthologs, providing insights into the molecular architecture of eIF5A and deoxyhypusine synthase (DHS) and their interaction. Contrary to previous suggestions of bifunctionality, our analyses indicate that the putative hydroxylation site in tvDHS is nonfunctional, and biochemical assays demonstrate exclusive deoxyhypusination capability. These findings challenge the notion of tvDHS functioning as both deoxyhypusine synthase and hydroxylase. The study enhances understanding of the hypusination pathway in T. vaginalis , shedding light on its functional relevance and potential as a drug target, and contributing to the development of novel therapeutic strategies against trichomoniasis.
The techniques of Microscale Thermophoresis (MST), and recently introduced Spectral Shift (SpS) have become valuable tools in target-based drug discovery owned to their ability of immobilization-free ...detection, low sample requirements, sensitivity, and scalability. These techniques allow fast detection and quantitative characterization of protein-ligand interactions and have found utility in library screening for hit compounds and characterization of interactions in the later stages of compound development. In this article, we highlight the advantages and limitations of MST and SpS in drug discovery and discuss how they can facilitate the characterization of new drug candidates. Our discussion is supported by case studies that demonstrate the successful application of the techniques in hit validation and optimization. In general, MST and SpS offer indispensable tools in drug discovery that support or even replace some of the earlier established approaches.
•Utility of Microscale Thermophoresis (MST) and Spectral Shift (SpS) in drug discovery is discussed.•Advantages and disadvantages of both approaches are clearly presented.•The methods are benchmarked against other popular screening/ligand binding characterizing approaches.•Case studies are highlighted which demonstrate the utility of the methods at various stages of drug discovery.
Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses ...the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved "ball-and-chain" motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.
Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The ...initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.
Proteasomes are responsible for protein turnover in eukaryotic cells, degrading short-lived species but also removing improperly folded or oxidatively damaged ones. Dysfunction of a proteasome ...results in gradual accumulation of misfolded/damaged proteins, leading to their aggregation. It has been postulated that proteasome activators may facilitate removal of such aggregation-prone proteins and thus prevent development of neurodegenerative disorders. However, the discovery of pharmacologically relevant compounds is hindered by insufficient structural understanding of the activation process. In this study we provide a model peptidic activator of human proteasome and analyze the structure-activity relationship within this novel scaffold. The binding mode of the activator at the relevant pocket within the proteasome has been determined by X-ray crystallography. This crystal structure provides an important basis for rational design of pharmacological compounds. Moreover, by providing a novel insight into the proteasome gating mechanism, our results allow the commonly accepted model of proteasome regulation to be revisited.
Glycerol is an organic compound that can be utilized as an alternative source of carbon by various organisms. One of the ways to assimilate glycerol by the cell is the phosphorylative catabolic ...pathway in which its activation is catalyzed by glycerol kinase (GK) and glycerol-3-phosphate (G3P) is formed. To date, several GK crystal structures from bacteria, archaea, and unicellular eukaryotic parasites have been solved. Herein, we present a series of crystal structures of GK from
(CtGK) in apo and glycerol-bound forms. In addition, we show the feasibility of an ADP-dependent glucokinase (ADPGK)-coupled enzymatic assay to measure the CtGK activity. New structures described in our work provide structural insights into the GK catalyzed reaction in the filamentous fungus and set the foundation for understanding the glycerol metabolism in eukaryotes.
The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin‐related modifier 1 (Urm1) pathway is responsible for ...the synthesis of 2‐thiolated wobble uridine (U34). During the key step of the modification cascade, the E1‐like activating enzyme ubiquitin‐like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C‐terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1‐COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin‐like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin‐like proteins (UBL) and sulfur‐relay systems. Here, we report the crystal structures of full‐length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C‐terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self‐conjugation with its own product, namely activated Urm1‐COSH.
Synopsis
tRNA thiolation factors Urm1‐Uba4 represent the most ancestral known ubiquitin‐like protein/E1 enzyme conjugation system, whose structural mechanisms have however remained elusive. Here, crystal structures and complementary biochemical and cellular analysis of fungal Urm1‐Uba4 show that it combines features of bacterial sulfur‐relay pathways and eukaryotic ubiquitin conjugation systems.
Full‐length Uba4 represents a non‐canonical E1 with an unexpected asymmetric domain architecture.
The Uba4 interdomain linker region has a critical role, and Uba4 adenylation activity is required to recruit its substrate Urm1.
The Uba4‐Urm1 complex structure provides molecular insights into substrate recognition, adenylation, and thiocarboxylation.
The catalytic cysteine responsible for E1 thioester formation protects Uba4 from its own product, Urm1‐COSH.
The covalent E1‐UBL self‐conjugation reaction observed in vitro might represent the root of all eukaryotic E1‐E2-E3 based UBL conjugation pathways.
Structural insights into the most ancestral known ubiquitin‐like protein/E1 enzyme conjugation system shows it to combine features of bacterial sulfur‐relay pathways and eukaryotic ubiquitin conjugation systems.
Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage ...assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium
Thermotoga maritima
(TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like
Archeoglobus fulgidus
ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg
2+
). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.
Modifications to a protein cage whose assembly depends on the presence of metal ions can modulate the extent of its dependence and in some cases convert the assembly to be salt independent.
ADP‐dependent glucokinase (ADPGK) is an alternative novel glucose phosphorylating enzyme in a modified glycolysis pathway of hyperthermophilic Archaea. In contrast to classical ATP‐dependent ...hexokinases, ADPGK utilizes ADP as a phosphoryl group donor. Here, we present a crystal structure of archaeal ADPGK from Methanocaldococcus jannaschii in complex with an inhibitor, 5‐iodotubercidin, d‐glucose, inorganic phosphate, and a magnesium ion. Detailed analysis of the architecture of the active site allowed for confirmation of the previously proposed phosphorylation mechanism and the crucial role of the invariant arginine residue (Arg197). The crystal structure shows how the phosphate ion, while mimicking a β‐phosphate group, is positioned in the proximity of the glucose moiety by arginine and the magnesium ion, thus providing novel insights into the mechanism of catalysis. In addition, we demonstrate that 5‐iodotubercidin inhibits human ADPGK‐dependent T cell activation‐induced reactive oxygen species (ROS) release and downstream gene expression, and as such it may serve as a model compound for further screening for hADPGK‐specific inhibitors.
PDB Code(s): 5OD2;
In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate. In contrast to canonical hexokinases, ...which use ATP, ADP-dependent glucokinase (ADPGK) catalyzes noncanonical phosphorylation of glucose to glucose 6-phosphate using ADP as a phosphate donor. Initially discovered in Archaea, the human homolog of ADPGK was described only recently. ADPGK's involvement in modified bioenergetics of activated T cells has been postulated, and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood, and effective ADPGK inhibitors still await discovery. Here, we show that 8-bromo–substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution, we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the protein's active site by engaging crucial catalytic residues. The results obtained using the Jurkat model of activated human T cells suggest its moderate activity in a cellular setting. We propose that our structural insights provide a critical basis for rational development of novel ADPGK inhibitors.
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