Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.
Lantibiotics are (methyl)-lanthionine-containing antimicrobial peptides produced by several Gram-positive bacteria. Some human pathogenic bacteria express specific resistance proteins that counteract this antimicrobial activity of lantibiotics. In Streptococcus agalactiae COH1 resistance against the well-known lantibiotic nisin is conferred by, the nisin resistance protein (NSR), a two-component system (NsrRK) and a BceAB-type ATP-binding cassette (ABC) transporter (NsrFP). The present study focuses on elucidating the function of NsrFP via its heterologous expression in Lactococcus lactis. NsrFP is able to confer a 16-fold resistance against wild type nisin as determined by growth inhibition experiments and functions as a lantibiotic exporter. Several C-terminal nisin mutants indicated that NsrFP recognizes the N-terminal region of nisin. The N-terminus harbors three (methyl)-lanthionine rings, which are conserved in other lantibiotics.
Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3' of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.
Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.
Lantibiotics are a growing class of natural compounds, which possess antimicrobial activity against a broad range of Gram-positive bacteria. Their high potency against human pathogenic strains such as MRSA and VRE makes them excellent candidates as substitutes for classic antibiotics in times of increasing multidrug resistance of bacterial strains. New lantibiotics are detected in genomes and can be heterologously expressed. The functionality of these novel lantibiotics requires a systematic purification and characterization to benchmark them against for example the well-known lantibiotic nisin. Here, we used a standardized workflow to characterize lantibiotics consisting of six individual steps. The expression and secretion of the lantibiotic was performed employing the promiscuous nisin modification machinery. We mutated the first amino acid of nisin into all proteinaceous amino acids and compared their bactericidal potency against sensitive strains as well as strains expressing nisin resistance proteins. Interestingly, we can highlight four distinct groups based on the residual activity of nisin against sensitive as well as resistant L. lactis strains.
Abstract Protein secretion is indispensable for essential cellular processes in eukaryotic cells, contributing significantly to nutrient acquisition, defense or communication. Alternative pathways bypassing the endomembrane system collectively referred to as unconventional secretion are gaining increasing attention. A number of important molecules such as cytokines, fibroblast growth factor or viral proteins are being exported through these mechanistically diverse pathways. In the fungal model Ustilago maydis , cytokinesis-dependent unconventional secretion mediates export of the chitinase Cts1 via the fragmentation zone. This membrane-rich compartment is formed during cytokinesis between mother and daughter cells. Recently, we identified Jps1, a previously uncharacterized protein, as a crucial factor for Cts1 localization and export. Combining biochemical experiments and in vivo studies, we here uncover two pivotal features of Jps1: dimerization and phosphoinositide (PIP) binding. Our findings reveal that a conserved structural core domain mediates homodimerization, while surrounding flexible variable regions suggest potential diversification in different basidiomycete species. Jps1 does not harbor a canonical PIP-binding domain but instead specificity of the interaction with the preferred PIP PI(4,5)P 2 is determined by basic residues. Importantly, loss of PI(4,5)P 2 binding specificity results in mis-localisation, morphological defects and reduced extracellular Cts1 activity, particularly at low cell densities. This discovery sheds light on previously unknown key features of Jps1, elucidating its role in supporting Cts1 secretion, and representing a crucial step towards understanding the broader implications of unconventional secretion in eukaryotic cells.
Heat shock protein 90 (Hsp90) is a promising therapeutic target due to its involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby contributing in exerting anti-apoptotic effects, which is essential for the malignant transformation and progression of several tumor types. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain (NTD) of Hsp90. However, adverse effects, including induction of the pro-survival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded clinical approval of these Hsp90i. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR and, thus, emerge as a promising alternative approach to target Hsp90. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with this essential dimerization process by small-molecule protein-protein interaction (PPI) inhibitors is a promising strategy for anticancer drug research. We have developed the first-in-class small molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitors (TKIs) resistant leukemia cells, without inducing HSR.
Abstract Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa . The lipase undergoes folding and activation in the periplasm prior the secretion. Here, we demonstrate that the ubiquitous periplasmic chaperone Skp of P. aeruginosa , but not SurA, FkpA, PpiD or YfgM, efficiently prevents misfolding of the aggregation-prone lipase A and facilitates its activation by a specific foldase LipH. Small-angle X-ray scattering visualizes the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 μM, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by high salt concentrations typical for the sputum of cystic fibrosis patients. The chaperoning effect of Skp points to its potent role in maturation and secretion of the lipase in Pseudomonas species.
Abstract To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell’s plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host’s endocytic system for efficient internalization.
Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied examples is nisin produced by Lactococcus lactis. Nisin is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT that secretes nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the nisin maturation machinery, consisting of the dehydratase NisB and the cyclase NisC. Our determined apparent secretion rates of NisT show how NisB and NisC modulate the transport kinetics of NisA. Additional in vitro studies of the detergent-solubilized NisT revealed how these enzymes and the substrates again influence the activity of transporter. In summary, this study highlights the pivotal role of NisB for NisT in the secretion process.
