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Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.
Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution prior to protein binding, and by release of these waters, the favored entropic binding is observed.
The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit ß of ribonucleotide reductase in Mesoplasma florum upon 2´-deoxyguanosine binding. We characterized binding of 2´-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and ‘in-cell-like’ conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. ‘In-celllike’ environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under ‘in-cell-like’-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2´-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2´-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2´-deoxyguanosine, guanosine plays a role in riboswitch function in vivo. Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2´ -deoxyguanosine was not able to regulate gene expression.
Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.
The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR),[1, 2] a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors.
The impact of the incorporation of a non-natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post-translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X-ray crystallography. The structure and dynamics of the apo state of AZH-modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH-modified PDZ3 and an azulene-linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene-linked peptide and the isotopically labelled protein. Co-crystallisation and soaking failed for the peptide-bound holo complex. NMR spectroscopy, however, allowed determination of the protein-ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA-modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target.
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2O2/NaHCO3, mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.
Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades.