When Wnt ligands bind to Frizzled (FZD) receptors, Dishevelled protein (DVL) gets multiphosphorylated by Casein kinase 1 (CK1). Although, it is well known that DVL phosphorylation relays Wnt signals from receptors to downstream effectors, any mechanistic aspects of DVL function related to phosphorylation remain unresolved. Here, we uncovered a Wnt-induced DVL phospho-switch which is mutually exclusive with FZD engagement. CK1 multiphosphorylation changes dramatically the bulk electrostatics to promote DVL intramolecular coupling between the DEP domain and the adjacent disordered region. A panel of phospho-switch mutants demonstrated a switch-like coupling at the molecular level when a charge threshold is reached. Charge accumulation proximal to DEP proved to be a key functional event required, but not sufficient, for Wnt/β-catenin signaling. Interestingly, the charge-dependent switch-like character of DVL controlled its association with FZD. By integrating findings at different levels, we propose a universal mechanism in which Wnt-induced DVL conformational phospho-switch outcompetes FZD binding and triggers DVL detachment from FZD. ### Competing Interest Statement The authors have declared no competing interest. The Czech Science Foundation (GACR), GA23-06913S, GA22-25365S Ministry of Education Youth and Sports, LUAUS25170, LX22NPO5102

One of the largest domain-motif interactomes in human involves PSD-95/Discs-large/ZO-1 (PDZ) domains. The framework for understanding the PDZ interactome is well established; however the functional dynamics associated with PDZ-ligand interactions are poorly understood. Here, we report a dual PDZ-binding mode that ascribes unique dynamic features to class III ligand recognition. The crystal structure revealed that the PDZ domain can recognize either of the carboxylate moieties (terminal or internal) present in the class III ligand and laid out the register rules responsible for the dual recognition. Variants of the ligand designed to retain one or the other carboxylate of the native sequence were sufficient for PDZ binding. The conformational dynamics of PDZ probed by NMR relaxation dispersion experiments demonstrated that the class III ligand is shuffling binding modes as it engages with the PDZ domain. Our mechanistic findings reveal yet another aspect of PDZ binding plasticity specific to class III ligands.

The PDZ domain of Dishevelled 3 protein belongs to a highly abundant protein recognition motif which typically binds short C-terminal peptides. The affinity of the PDZ towards the peptides could be fine-tuned by a variety of post-translation modifications including phosphorylation. However, how phosphorylations affect the PDZ structure and its interactions with ligands remains elusive. Combining molecular dynamics simulations, NMR titration, and biological experiments, we explored the role of previously reported phosphorylation sites and their mimetics in the Dishevelled PDZ domain. Our observations suggest three major roles for phosphorylations: (1) acting as an on/off PDZ binding switch, (2) allosterically affecting the binding groove, and (3) influencing the secondary binding site. Our simulations indicated that mimetics had similar but weaker effects, and the effects of distinct sites were non-additive. This study provides insight into the Dishevelled regulation by PDZ phosphorylation. Furthermore, the observed effects could be used to elucidate the regulation mechanisms in other PDZ domains.

The Wnt signaling pathway is linked to multiple developmental defects, inherited diseases, and many types of cancer. An essential component of the Wnt signaling pathway is a family of proteins known as Dishevelled (DVL). Studies have hinted that phosphorylation of DVL proteins at specific sites ultimately controls parts of the Wnt pathway, but the molecular mechanisms have remained unclear. Now, researchers have discovered “barcode”-like patterns of phosphorylation that could dictate certain DVL functions. The team used mass spectrometry-based proteomics to determine which sites along the protein DVL3 were phosphorylated by 8 different kinases and found 88 phosphorylation sites organized into barcodes unique for each kinase. The barcoding they observed could determine how DVL3 is distributed in the cell and, with further research, could point to the role of DVL proteins in certain human diseases and cancers.

By understanding the rampant infections of Mycobacterium tuberculosis (Mtb) and inflammations caused due to the generation of radical species during the Mtb infection, a series of (E)-2-(2-allylidenehydrazinyl)thiazole derivatives, with dual-action properties, was designed. The molecules were designed with a considerable variation in LogP, one of the critical parameters in physicochemical properties, and analyzed for their drug-likeness. For the synthesis, a simple, green, and multicomponent one-pot synthesis method was developed. The in vitro inhibition potentials were evaluated against Mtb H37 Rv by the microplate Alamar Blue assay. The results reveal that compound 6 was potent, with a MIC value of 6.5 µg/ml, and showed better interactions with the KasA protein with binding free energy (ΔG) of -9.4 kcal/mol. Also, the radical scavenging properties were studied to establish the dual-action properties of the molecules. Compound 9 exhibited promising antioxidant and nitric oxide radical scavenging activities, with 81.7% and 81.0%, respectively, at 1,000-μg/ml concentration.

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.

Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630–643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome.

The reductive discoloration of azo dye, Congo red, catalyzed by noble metal nanoparticles was used to visualize protein-metal complexes in native polyacrylamide gels after counterstaining with Coomassie blue. This technique was used to characterize the synthesis of palladium, rhodium and iridium nanoparticles encapsulated in Pyrococcus furiosus ferritin.

Experimental Section Cloning, expression, and preparation of Pyrococcus ferritin: The open reading frame (ORF) of Pyrococcus furiosus ferritin (PfuFt) gene DSM3638 strain was codon optimized for expression in E. coli K12 (GeneOptimizer, Thermo Fisher Scientific). The PfuFt ORF in the recombinant protein is preceded by Met-Ala in order to introduce NcoI site in-frame for cloning purposes. The gene synthesis and cloning into pSFT7 plasmid were carried out by Genewiz. The coding sequence was cloned into pET28(a) bacterial expression plasmid. The protein was produced in BL21-CodonPlus (DE3)-RIL (Agilent) E. coli strain induced with 0.5 mM IPTG. Induced bacteria were collected by centrifugation and the pellet resuspended in Ft lysis buffer (FLB) (25 mM 1/2Na.HEPES, 150 mM NaCl). Bacteria were lysed by sonication, centrifuged and the clarified lysate was heated for 15 min to 80 °C in order to denature mesophilic proteins that were not removed by centrifugation. Cleared supernatant was further treated with DNaseI (10 μg/ml) for 3 h at 37 °C in order to remove bacterial DNA. DNaseI was removed by second heat treatment (10 min at 90 °C). PfuFt was concentrated from clarified heat-treated samples by filtration through Amicon Ultra-2 (Ultracel – 100K membrane with exclusion limit 100 000 NMWL) (Millipore). Low molecular weight contaminants were removed by size exclusion chromatography with PD MiniTrap G-25 (GE Healthcare) equilibrated with FLB. The protein assembly and purity were analyzed by native and denaturing polyacrylamide gel electrophoresis.

Comparative mode of action of some terpene compounds against octopamine receptor and acetyl cholinesterase of mosquito and human system by the help of homology modeling and Docking studiesBulbuli Khanikor, Pratap Parida, R.N.S. Yadav and Dipsikha Bora

Cysteine proteinases are required for a wide range of physiological processes in all living organisms. In parasitic nematodes, they are particularly crucial for the digestion of host tissues and evasion of host immune responses. Therefore, in general, these are identified as primary targets for the control of parasitic nematodes. Herein, cathepsin S-like cysteine proteinase of Heterodera avenae (Hacp-s) has been cloned and analysed for the first time. The predicted protein is 298 amino acids long and showed significant similarity with cathepsin S of Heterodera glycines (Hgcp-s). The sequence of cathepsin S contains a signal peptide of 30 amino acids which suggests its role in extracellular functions. Multiple sequence alignment revealed the presence of ERFNIN motif and conserved catalytic residues. Three dimensional structure (3D) of Hgcp-s was modelled using homology modelling. In order to illustrate the plausible mode of interaction of cathepsin S (Hgcp-s), docking analysis was performed with E-64 cysteine proteinase inhibitor. Docking studies revealed the hydrogen bonding of E-64 with Gln153, His299 and Gly203 as well as close interaction with catalytic residues Cys159 and Asn320 Expression analysis of Hacp-s using qRT-PCR showed high expression of cathepsin S in pre parasitic J2s and female stages suggesting its significant role in both pre-parasitic and parasitic stages of the nematode life cycle.

BACKGROUND:New Delhi metallo-β-lactamase-1 (NDM-1)-producing Gram-negative bacteria are today's major worldwide health concern. The enzyme NDM-1 provides bacterial resistance by its hydrolytic activity against the β-lactam ring of antibiotics. Inhibition of NDM-1 may prevent the hydrolysis of β-lactam ring of the antibiotics, and therefore, plays an important role against antibacterial resistance.MATERIALS AND METHODS:Here we made an attempt to design suitable inhibitors against NDM-1 from different natural antibacterial compounds using molecular docking approach.RESULTS:We observed that natural compounds such as Nimbolide and Isomargololone are showing an appreciable IC50 value as well as significant binding energy value for NDM-1. We further observed these compounds showing better affinity to NDM-1 on comparison with 14 β-lactam antibiotics.CONCLUSION:Finally, our study provides a platform for the development of a potent inhibitor of NDM-1, which may be considered as a potential drug candidate against bacterial resistance.