Proteins with intrinsically disordered domains are implicated in a vast range of biological processes, especially cell signaling and regulation. Having solved the quaternary structure folded tumor suppressor p53 by multidisciplinary approach, we have now determined average ensemble N-terminal transactivation domain (TAD) using residual dipolar couplings (RDCs) from NMR spectroscopy small-angle x-ray scattering (SAXS). Remarkably, not only were able to measure RDCs isolated TAD, but also do...

The tumor suppressor p53 is mutationally inactivated in approximately 50% of human cancers. Approximately one-third the mutations lower melting temperature protein, leading to its rapid denaturation. Small molecules that bind those mutants and stabilize them could be effective anticancer drugs. mutation Y220C, which occurs 75,000 new cancer cases per annum, creates a surface cavity destabilizes protein by 4 kcal/mol, at site not functional. We have designed series binding from an silico...

Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. receptors target cargo to autophagy binding ATG8 on autophagosomal membranes. The expansion of the family in higher eukaryotes suggests that specific interactions with facilitate differential handling. However, selective interactors orthologs are unknown. Here we show selectivity receptor NDP52 LC3C is crucial innate immunity since cells lacking either protein cannot...

The destabilizing p53 cancer mutation Y220C creates a druggable surface crevice. We developed strategy exploiting halogen bonding for lead discovery to stabilize the mutant with small molecules. designed halogen-enriched fragment libraries (HEFLibs) as starting points complement classical approaches. From screening of HEFLibs and subsequent structure-guided design, we substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols p53-Y220C stabilizers. Crystal structures their complexes highlight two...

Wnt/β-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, β-catenin is targeted continuously for proteasomal degradation by Axin destruction complex, whose activity blocked upon Wnt stimulation Dishevelled, which recruits to plasma membrane assembles it into a signalosome. This key event during signal transduction depends on dynamic head-to-tail polymerization DIX domain Dishevelled. Here, we...

The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate by lowering its stability. We determined structure in solution using state-of-the-art isotopic labeling techniques NMR spectroscopy complement crystal structure. was very similar but far more mobile than expected. Importantly, we were able analyze structural environment several buried polar groups, indicated...

Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which Dishevelled via reversible head-to-tail polymerization its DIX domain. Its activity causes stabilization of β-catenin, a effector with pivotal roles in animal development and cancer. How triggers signalosome assembly unknown. Here, we use structural...

Wnt/β-catenin signalling controls development and tissue homeostasis. Moreover, activated β-catenin can be oncogenic and, notably, drives colorectal cancer. Inhibiting has proven a formidable challenge. Here we design screen for small-molecule inhibitors of β-catenin's binding to its cofactor BCL9, discover five related natural compounds, including carnosic acid from rosemary, which attenuates transcriptional outputs in cancer cells. Evidence NMR analytical ultracentrifugation demonstrates...

The p53 cancer mutation Y220C induces formation of a cavity on the protein's surface that can accommodate stabilizing small molecules. We combined fragment screening and molecular dynamics to assess druggability p53-Y220C map ligand interaction sites within mutational cavity. Elucidation binding mode hits by crystallography yielded clear picture how drug might dock in Simulations solvate protein with isopropanol found additional extend druggable surface. Moreover, structural observations...

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free thermally unstable protein that equilibrium between monomers dimers. Self-association modulated by basic C-terminal tail. The DNA-binding ability mainly contributed first HMG-box, while the second HMG-box has low-DNA-binding...

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to Pygo chromatin reader acting through TCF/LEF-responsive enhancers. renders this Wnt-responsive, capturing β-catenin via Legless/BCL9 adaptor. We used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) human BCL9 B9L show C-terminus downstream their adaptor elements is crucial for...

ABSTRACT Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces signals to cytoplasmic effectors including β-catenin, control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies relied on Wnt-independent signalling activity resulting from overexpression, which is sufficient trigger polymerisation, bypassing the requirement for signals. Here, we generate a...

p53 binds to some members of the S100 family (S100B, S100A4, S100A2, and S100A1). We previously showed that both S100B S100A4 bind tetramerization domain, consequently control its oligomerization state, but only C-terminal negative regulatory domain (NRD). Here, we investigate other binding partners for within (S100A6 S100A11), show seems be a general feature family, while NRD is characteristic subset family.

Oncogenic mutations inactivate the tumor suppressor p53 by lowering its stability or weakening binding to DNA. Alkylating agents that reactivate mutant are currently being explored for cancer therapy. We have discovered ligands containing an α,β-unsaturated double bond, characteristic of Michael acceptors, bind covalently generic cysteine sites in core domain. They raised melting temperature domain wild-type and hotspot mutants R175H, Y220C, G245S, R249S, R282 up 3°C. Analysis relative...

The proteins MDM2 and MDM4 are key negative regulators of the tumor suppressor protein p53, which frequently upregulated in cancer cells. They inhibit transactivation activity p53 by binding separately or concert to its domain. is also a ubiquitin ligase that leads degradation p53. Accordingly, important targets for drugs their We found from silico screening confirmed experiment lithocholic acid (LCA) binds sites both with fivefold preference MDM4. LCA an endogenous steroidal bile acid,...

Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They built around central cartwheel assembly organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate thereby centriole symmetry unclear. Here we show Leishmania major crystallizes as 9-fold provide X-ray structure this at resolution 3.5 Å. We furthermore demonstrate oligomerization be...

The Pygo-BCL9 complex is a chromatin reader, facilitating β-catenin-mediated oncogenesis, and thus emerging as potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygo's PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from BCL9 HD1 domain. Here, we report first use fragment-based screening by NMR to identify small molecules block protein-protein interactions finger. This led discovery set benzothiazoles...

TCF/LEF factors are ancient context-dependent enhancer-binding proteins that activated by β-catenin following Wnt signaling. They control embryonic development and adult stem cell compartments, their dysregulation often causes cancer. β-catenin-dependent transcription relies on the NPF motif of Pygo proteins. Here, we use a proteomics approach to discover Chip/LDB-SSDP (ChiLS) complex as ligand specifically binding NPF. ChiLS also recognizes motifs in other nuclear including Runt/RUNX2...

FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, translocates double-stranded until both dif recombination sites are placed at mid for subsequent Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of domain while translocating on its substrate. Each subunit homo-hexameric ring adopts unique conformation and one three nucleotide states. Two DNA-binding loops within four subunits form pair spiral...

A detailed investigation has been undertaken on the extent and nature of torsional fluctuations about glycosidic linkage model disaccharide Man alpha 1-3Man 1-OMe. In particular, we sought to determine whether three nuclear Overhauser effects two long-range heteronuclear 3JCH spin coupling constants measurable across were consistent with a single conformation or multiple conformations that linkage. Within experimental error, have found these five parameters can be interpreted in terms...

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild hydrolysis conditions that preferentially cleave phosphodiester linkages. saccharides shown to consist monomeric mannose and series neutral phosphorylated by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions,...