53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling.

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Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of (19)F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, (19)F spin-lattice relaxation rates, (1)H-(19)F nuclear Overhauser effects, and paramagnetic shifts and relaxation rates from dissolved oxygen. Both the solvent isotope shifts and paramagnetic shifts from dissolved oxygen sensitively reflect solvent exposed surface areas.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes.DOI: http://dx.doi.org/10.7554/eLife.23872.001

Although many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, ¹⁹F NMR and near-UV circular dichroism (CD) are used to characterize a transition to a thermal folding intermediate of calmodulin, a water-soluble protein, which is biosynthetically enriched with 3-fluorophenylalanine (3F-Phe). ¹⁹F NMR solvent isotope shifts, resulting from replacing H₂O with D₂O, and paramagnetic shifts arising from dissolved O₂ are used to monitor changes in the water accessibility and hydrophobicity of the protein interior as the protein progresses from a native state to an unfolded state along a heat-denaturation pathway. In comparison to the native state, the solvent isotope shifts reveal the decreased presence of water in the hydrophobic core, whereas the paramagnetic shifts show the increased hydrophobicity of this folding intermediate. ¹⁵N, ¹H and methyl ¹³C,¹H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure whose hydrophobic interior is highly dynamic. ¹⁹F NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.