Structural probing: The activity of thiostrepton and derivatives with targeted shape changes was determined at their ribosomal binding site by using semisynthesis, NMR structure determination, docking (see picture), and biological evaluation in an integrated fashion. This combination revealed important elements of molecular recognition within the embedded pharmocophore of the target, a composite RNA–protein complex.
Abstract Kupfer (Cu) ist ein Übergangsmetall, das eine entscheidende Rolle im Zellstoffwechsel spielt. Die Cu + ‐Homöostase ist in vielen Krebsarten hochreguliert und trägt zur Tumorentstehung bei. Therapeutische Strategien zur gezielten Beeinflussung der Cu + ‐Homöostase in Krebszellen werden jedoch selten erforscht, da kleine Cu + ‐Chelatoren eine geringere Bindungsaffinität aufweisen als intrazelluläre Cu + ‐Chaperone, Enzyme oder Liganden. Um dieses Problem anzugehen, stellen wir einen supramolekularen Ansatz vor, der von Cu + ‐Chaperonen inspiriert ist, um die Cu + ‐Homöostase in Krebszellen zu stören und somit den programmierten Zelltod zu induzieren. Das Peptid Nap‐FFMTCGGCR bildet in Krebszellen Nanofibrillen aus, die aufgrund des einzigartigen MTCGGC‐Motivs, das in intrazellulären Cu + ‐Chaperonen konserviert ist, eine hohe Bindungsaffinität und Selektivität für Cu + aufweisen. Nap‐FFMTCGGCR zeigt Zytotoxizität gegenüber dreifach negativen Brustkrebszellen (MDA‐MB‐231), beeinträchtigt die Aktivität des Cu + ‐abhängigen Co‐Chaperons Superoxid‐Dismutase 1 (SOD1) und induziert oxidativen Stress. Im Gegensatz dazu hat Nap‐FFMTCGGCR nur minimale Auswirkungen auf normale HEK 293T‐Zellen. Kontrollpeptide zeigen, dass Selbstassemblierung und Cu + ‐Bindung synergistisch wirken müssen, um die Cu + ‐Homöostase erfolgreich zu stören. Wir zeigen, dass die durch Assemblierung verstärkte Affinität für Metallionen neue therapeutische Strategien eröffnet, um krankheitsrelevante Metallionen‐Homöostase anzugehen.
SSR128129E (SSR) is a unique small‐molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high‐affinity allosteric binder that selectively blocks one of the two major FGFR‐mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N‐terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b‐induced FGFR signaling compared to FGF2‐induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N‐terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water‐soluble subdomains of the FGFR proteins points to the important role played by the cell membrane.
Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1−3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing the 1H−15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling.
Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application of PRE NMR on RNAs is a significant challenge due to the limited size of chemically synthesized RNA. Here, we present a noncovalent spin labeling strategy to spin label RNAs in high yields required for NMR studies. The approach requires the presence of a helix segment composed of about 10 nucleotides (nt) but is not restricted by the size of the RNA. We show successful application of this strategy on the 2′dG sensing aptamer domain of Mesoplasma florum (78 nt). The aptamer domain was prepared in two fragments. A larger fragment was obtained by biochemical means, while a short spin labeled fragment was prepared by chemical solid-phase synthesis. The two fragments were annealed noncovalently by hybridization. We performed NMR, cw-EPR experiments and gel shift assays to investigate the stability of the two-fragment complex. NMR analysis in 15N-TROSY and 1H,1H-NOESY spectra of both unmodified and spin labeled aptamer domain show that the fragmented system forms a stable hybridization product, is in structural agreement with the full length aptamer domain and maintains its function. Together with structure modeling, experimentally determined 1H-Γ2 rates are in agreement with reported crystal structure data and show that distance restraints up to 25 Å can be obtained from NMR PRE data of RNA.
Abstract The splicing isoform b of human fibroblast growth factor 8 (FGF8b) is an important regulator of brain embryonic development. Here, we report the almost complete NMR chemical shift assignment of the backbone and aliphatic side chains of FGF8b. Obtained chemical shifts are in good agreement with the previously reported X-ray data, excluding the N-terminal gN helix, which apparently forms only in complex with the receptor. The reported data provide an NMR starting point for the investigation of FGF8b interaction with its receptors and with potential drugs or inhibitors.
Target TAR by NMR: Tripeptides containing arginines as terminal residues and non-natural amino acids as central residues are good leads for drug design to target the HIV trans-activation response element (TAR). The structural characterization of the RNA-ligand complex by NMR spectroscopy reveals two specific binding sites that are located at bulge residue U23 and around the pyrimidine-stretch U40-C41-U42 directly adjacent to the bulge.
Abstract The energy landscapes of human telomeric G‐quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real‐time NMR spectroscopy, we investigated the K + ‐induced folding of the human telomeric DNA sequence 5′‐TTGGG(TTAGGG) 3 A‐3′. Three long‐lived states were detected during folding: a major conformation (hybrid‐1), a previously structurally uncharacterized minor conformation (hybrid‐2), and a partially unfolded state. The minor hybrid‐2 conformation is formed faster than the more stable hybrid‐1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin‐like structures.
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