scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Timothy S Strutzenberg ◽  
Ruben D Garcia-Ordonez ◽  
Scott J Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Small Molecules ◽  
Structural Dynamics ◽  
Site Directed Mutagenesis ◽  
Chemical Probes ◽  
Structural Determinants ◽  
X Ray ◽  
Synthetic Ligand ◽  
Hdx Ms

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it’s unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.

scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

2019 ◽  
Author(s):  
Timothy S. Strutzenberg ◽  
Ruben Garcia-Ordonez ◽  
Scott Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Orphan Nuclear Receptor ◽  
Chemical Probes ◽  
Structural Determinants ◽  
Synthetic Ligand ◽  
Hdx Ms

ABSTRACTMembers of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism1 to inflammation2 and cancer3. As ligand-gated transcription factors, synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology4. However, nearly half of NRs do not have specific cognate ligands or its unclear if they possess ligand dependent activities and these receptors are called orphans. Here we demonstrate that ligand-dependent action of the orphan nuclear receptor RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings are corroborated with X-ray co-crystallography and site-directed mutagenesis to collectively reveal the structural determinants of RORγ ligand-dependent activation, critical for designing full agonists for application in cancer immunotherapy. Combined these observations support a model of receptor activation to more accurately describe RORγ pharmacology. Likewise, this ‘bump-and-hole’ inspired approach could be extended to other orphan NRs to explore the ligand-dependent activities that are important for defining pharmacology.

scholarly journals Characterization of the Structural Determinants of the Ubiquitin-Dependent Proteasomal Degradation of Human Hepatic Tryptophan 2,3-Dioxygenase

2021 ◽  
Author(s):  
Yi Liu ◽  
Sung Mi Kim ◽  
YongQiang Wang ◽  
Shay Karkashon ◽  
Ariel Lewis-Ballester ◽  
...  
Keyword(s):  
Proteasomal Degradation ◽  
Site Directed Mutagenesis ◽  
Liver Protein ◽  
Lysosomal Degradation ◽  
Structural Determinants ◽  
The Poor ◽  
Structural Relevance ◽  
Positively Charged

Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ~2.3 h, relative to an average t1/2 of ~2–3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation (UPD) pathway. Additionally, we identified 15 ubiquitination K sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here we further established autophagic lysosomal degradation (ALD) as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

scholarly journals The Role of Percent Volume Buried in the Characterization of Copper(I) Complexes for Lighting Purposes

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2647 ◽  
Author(s):  
Murat Alkan-Zambada ◽  
Edwin C. Constable ◽  
Catherine E. Housecroft
Keyword(s):  
Solid State ◽  
Single Crystal ◽  
Quantum Yields ◽  
X Ray ◽  
Photophysical Behavior

The usefulness of percent volume buried (%Vbur) as a readily quantifiable property is investigated with regard to [Cu(NN)(PP)]+ complexes of interest for lighting purposes. Photoluminescence quantum yields (PLQYs) and single crystal X-ray structures of 100 reported compounds were assembled, %Vbur of the ligand systems were calculated and analyzed for correlations. We found that increased shielding of the central Cu(I) cation relying on shared contributions of both (NN) and (PP) ligand systems led to increased PLQYs. These findings are of relevance for future characterizations of Cu(I)-based complexes and their photophysical behavior in the solid-state.

scholarly journals Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states

2013 ◽  
Vol 69 (10) ◽  
pp. 2050-2060 ◽  
Author(s):  
Bodo Sander ◽  
Giancarlo Tria ◽  
Alexander V. Shkumatov ◽  
Eun-Young Kim ◽  
J. Günter Grossmann ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Cd Spectroscopy ◽  
X Ray ◽  
Force Microscopy ◽  
X Ray Scattering ◽  
Atomic Force ◽  
Extended States ◽  
High Degree

Gephyrin is a trimeric protein involved in the final steps of molybdenum-cofactor (Moco) biosynthesis and in the clustering of inhibitory glycine and GABAAreceptors at postsynaptic specializations. Each protomer consists of stably folded domains (referred to as the G and E domains) located at either terminus and connected by a proteolytically sensitive linker of ∼150 residues. Both terminal domains can oligomerize in their isolated forms; however, in the context of the full-length protein only the G-domain trimer is permanently present, whereas E-domain dimerization is prevented. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) reveal a high degree of flexibility in the structure of gephyrin. The results imply an equilibrium between compact and extended conformational states in solution, with a preference for compact states. CD spectroscopy suggests that a partial compaction is achieved by interactions of the linker with the G and E domains. Taken together, the data provide a rationale for the role of the linker in the overall structure and the conformational dynamics of gephyrin.

Growth and characterization of Al–Cu–Li quasicrystals

1988 ◽  
Vol 3 (2) ◽  
pp. 233-237 ◽  
Author(s):  
J. M. Parsey ◽  
H. S. Chen ◽  
A. R. Kortan ◽  
F. A. Thiel ◽  
A. E. Miller ◽  
...  
Keyword(s):  
Phase Diagram ◽  
Alloy System ◽  
Icosahedral Phase ◽  
Solidification Velocity ◽  
Scanning Calorimetry ◽  
X Ray ◽  
Pseudobinary Phase Diagram ◽  
Melt Composition

The alloy system Al–Li–Cu was investigated extensively over the composition range 5.8–8 Al–Cu–3Li to develop a detailed understanding of the formation and properties of the icosahedral phase, known as T2. Material from the various charges was analyzed by optical and electron microscopy, energy dispersive x-ray analysis, differential thermal analysis, and differential scanning calorimetry. The role of the melt composition and the solidification velocity were found to be crucial in determining the micro- and macrostructure and the existence of the icosahedral phase. A pseudobinary phase diagram for the region around Al6CuLi3, is presented based on these analyses. Based on this phase diagram the largest single icosahedral crystals of Al5.1CuLi3 yet reported, with diameters greater than 1 cm, were produced by Bridgman methods.

Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase

2018 ◽  
Vol 475 (24) ◽  
pp. 3963-3978 ◽  
Author(s):  
Gregory Arnal ◽  
Peter J. Stogios ◽  
Jathavan Asohan ◽  
Tatiana Skarina ◽  
Alexei Savchenko ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Glycoside Hydrolase ◽  
Site Directed Mutagenesis ◽  
Glycoside Hydrolase Family ◽  
X Ray ◽  
Recombinant Production ◽  
Structural Enzymology ◽  
Hydrophobic Binding ◽  
Hydrolase Family

Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (PoGH74cat) revealed a highly specific, processive endo-xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform — the first in GH74 extending from subsites −4 to +6 — and unique mobile active-site loops. Site-directed mutagenesis revealed that glycine-476 was uniquely responsible for the promiscuous backbone-cleaving activity of PoGH74cat; replacement with tyrosine, which is conserved in many GH74 members, resulted in exclusive hydrolysis at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity.

scholarly journals Synthesis of Iron Oxide Nanoparticles Using Isobutanol

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Diana Kostyukova ◽  
Yong Hee Chung
Keyword(s):  
Iron Oxide ◽  
Iron Oxide Nanoparticles ◽  
Ammonium Hydroxide ◽  
Oxide Nanoparticles ◽  
X Ray Diffraction ◽  
Ferrous Chloride ◽  
X Ray ◽  
Superparamagnetic Properties

Iron oxide nanoparticles were synthesized by precipitation in isobutanol with sodium hydroxide and ammonium hydroxide. The isobutanol played a role of a surfactant in the synthesis. The nanoparticles were calcined for 100 min to 5 hours in the range of 300 to 600°C. The characterization of the samples by FTIR (Fourier-transform infrared) and XRD (X-ray diffraction) confirmed the formation ofγ-Fe2O3(maghemite) from Fe3O4(magnetite) at calcination at 300°C. The morphology and particle size were studied by SEM (scanning electron microscope). Nanoparticles in the range of 11–22 nm prepared at 0.09 M of ferrous chloride exhibited superparamagnetic properties. Nanoparticles synthesized with ferrous chloride and ammonium hydroxide at 75°C and calcined at 530°C for 2 h wereα-Fe2O3(hematite).

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