The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)-DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4' of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key DNA repair enzyme that excises 8-oxoguanine, a mutagenic base lesion, from double-stranded DNA. In this study, we crystallographically visualized an intermediate state of the enzymatic reaction. To achieve this, we employed a specifically designed pH-sensitive mutant of hOGG1 and applied a freeze-trapping technique to capture the reaction intermediate. The resulting crystal structure revealed a previously unknown reaction pathway involving a hemiaminal-type intermediate, captured here for the first time. These findings provide new insights into the catalytic mechanism of hOGG1.

• MeSH
• DNA-glykosylasy * chemie   genetika   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• guanin analogy a deriváty   chemie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 8-hydroxyguanine MeSH Prohlížeč
• DNA-glykosylasy * MeSH
• DNA MeSH
• guanin MeSH
• oxoguanine glycosylase 1, human MeSH Prohlížeč

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein.

• Klíčová slova
• anti-cancer therapy, nuclear magnetic resonance, protein-peptide docking, protein-protein interactions, small-molecule inhibitors,
• MeSH
• alternativní sestřih * MeSH
• lidé MeSH
• ligandy MeSH
• magnetická rezonanční spektroskopie MeSH
• počítače MeSH
• spliceozomy * metabolismus   MeSH
• ubikvitiny genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• ubikvitiny MeSH

Nuclear magnetic resonance (NMR) spectroscopy is a key method for determining the structural dynamics of proteins in their native solution state. However, the low sensitivity of NMR typically necessitates nonphysiologically high sample concentrations, which often limit the relevance of the recorded data. We show how to use hyperpolarized water by dissolution dynamic nuclear polarization (DDNP) to acquire protein spectra at concentrations of 1 μM within seconds and with a high signal-to-noise ratio. The importance of approaching physiological concentrations is demonstrated for the vital MYC-associated factor X, which we show to switch conformations when diluted. While in vitro conditions lead to a population of the well-documented dimer, concentrations lowered by more than two orders of magnitude entail dimer dissociation and formation of a globularly folded monomer. We identified this structure by integrating DDNP with computational techniques to overcome the often-encountered constraint of DDNP of limited structural information provided by the typically detected one-dimensional spectra.

• Publikační typ
• časopisecké články MeSH

We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2'-deoxyguanosine to the aptamer domain of the bacterial 2'-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.

• Klíčová slova
• 2′-deoxyguanosine riboswitch, HeLa cells, RNA structures, aptamers, structural biology,
• MeSH
• aptamery nukleotidové chemie   metabolismus   MeSH
• HeLa buňky MeSH
• konfokální mikroskopie MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• ligandy MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• riboswitch MeSH
• RNA chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aptamery nukleotidové MeSH
• ligandy MeSH
• riboswitch MeSH
• RNA MeSH

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

• Klíčová slova
• 3D NMR, BEST-HNCO, Direct 13C detection, Dissolution-dynamic nuclear polarization (D-DNP), Hyperpolarization, Non-uniform sampling,
• MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• osteopontin chemie   MeSH
• ubikvitin chemie   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• osteopontin MeSH
• SPP1 protein, human MeSH Prohlížeč
• ubikvitin MeSH
• vnitřně neuspořádané proteiny MeSH
• voda MeSH