Background Hyper-IgE Syndrome (HIES) is a rare, autosomal dominant (AD) immunodeficiency characterized by eczema, Staphylococcus aureus skin abscesses, pneumonia with pneumatocele formation, Candida infections, and skeletal/connective tissue abnormalities. Recently it was shown that heterozygous STAT3 mutations cause AD-HIES. Objective To determine the spectrum and functional consequences of heterozygous STAT3 mutations in a cohort of HIES patients. Methods We sequenced the STAT3 gene in 38 HIES patients (NIH-score >40 points) from 35 families, quantified TH17 cells in peripheral blood, and evaluated tyrosine phosphorylation of STAT3. Results Most STAT3 mutations in our cohort were in the DNA-binding domain (DBD) (22/35 families) or SH2 domain (10/35), and were missense mutations. We identified two intronic mutations resulting in exon skipping and in-frame deletions within the DBD. In addition, we identified two mutations located in the transactivation domain downstream of the SH2 domain: A ten amino acid deletion and an amino acid substitution. In one patient, we were unable to identify a STAT3 mutation. TH17 cells were absent or low in the peripheral blood of all patients who were evaluated (n=17). IL-6 induced STAT3-phosphorylation was consistently reduced in patients with SH2 domain mutations, but comparable to normal controls in patients with mutations in the DBD. Conclusion Heterozygous STAT3 mutations were identified in 34/35 unrelated HIES families. Patients had impaired TH17 cell development, and those with SH2 domain mutations had reduced STAT3 phosphorylation. Clinical implication Mutations in STAT3 and decreased TH17 cells identify individuals with AD-HIES, thereby allowing timely diagnosis and early treatment of these patients. Capsule summary Results from this patient cohort expand the spectrum of heterozygous STAT3 mutations in AD-HIES, and demonstrate impaired development of TH17 cells in all and reduced STAT3-phosphorylation in patients with SH2-domain mutations.
An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large‐amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (<milliseconds). Here, we review RDC methodology for studying RNA structural dynamics and survey what has been learnt thus far from application of these methods. Future methodological challenges are also identified. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 384–402, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.
The P4 helix is an essential element of ribonuclease P (RNase P) that is believed to bind catalytically important metals. Here, we applied a combination of NMR residual dipolar couplings (RDCs) and a recently introduced domain-elongation strategy for measuring ''motionally decoupled'' relaxation data to characterize the structural dynamics of the P4 helix from Bacillus subtilis RNase P. In the absence of divalent ions, the two P4 helical domains undergo small amplitude (;13°) collective motions about an average interhelical angle of 10°. The highly conserved U7 bulge and helical residue C8, which are proposed to be important for substrate recognition and metal binding, are locally mobile at pico-to nanosecond timescales and together form the pivot point for the collective domain motions. Chemical shift mapping reveals significant association of Mg 2+ ions at the P4 major groove near the flexible pivot point at residues (A5, G22, G23) previously identified to bind catalytically important metals. The Mg 2+ ions do not, however, significantly alter the structure or dynamics of P4. Analysis of results in the context of available Xray structures of the RNA component of RNase P and structural models that include the pre-tRNA substrate suggest that the internal motions observed in P4 likely facilitate adaptive changes in conformation that take place during folding and substrate recognition, possibly aided by interactions with Mg 2+ ions. Our results add to a growing view supporting the existence of functionally important internal motions in RNA occurring at nanosecond timescales.
Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn 2þ bound to a P4 helix mimic is sixcoordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn 2þ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with CoðNH 3 Þ 3þ 6 , Zn 2þ , and CoðNH 3 Þ 3þ 6 ∕Zn 2þ confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.manganese | ribozyme | RNase P | X-ray absorption spectroscopy | zinc R NA molecules are large polyanions that associate with numerous divalent metal ions that stabilize their structure and promote catalysis. Identification of the RNA moieties involved in metal-binding and discerning the nature of these interactions is an important outstanding question in the field (1, 2). Whereas RNA-bound metals can be characterized at the atomic level in the solid-state by X-crystallography there are not yet techniques for characterizing the binding sites and coordination schemes for RNA-bound metals in solution. Techniques based on NMR, EPR, and Raman spectroscopy as well as nucleotide analog interference mapping (NAIM) and phosphorothioate substitution exist for identifying RNA residues involved in metal-binding; however the geometry of the bound metal and the nature of the coordination scheme cannot be resolved based on these experiments alone.RNase P is a metal-dependent ribozyme that catalyzes precursor tRNA (pre-tRNA) maturation by cleaving a specific phosphodiester bond (3). In RNase P, metal ions stabilize the folded RNase P RNA (PRNA) structure, enhance ligand affinity, and stabilize the transition state for cleavage (4); in vivo the metal requirement is fulfilled by Mg 2þ ions (5, 6). A majority of the ∼150 divalent metal ions associated with PRNA bind nonspecifically via electrostatic interactions whereas only a handful of ions form specific contacts with RNase P (5, 7). Discerning the position and structure of the few divalent ions that site-specifically interact with RNA is a majo...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.