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.

Spinach is an in vitro selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence.Spinach is thus an RNA analog of GFP, and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2 and 2.4 Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially pre-formed binding site for the fluorophore.The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

A complex interplay of signaling events, including the Wnt pathway, regulates sprouting of blood vessels from preexisting vasculature during angiogenesis. Here we show that two distinct mutations in the (uro)chordate-specific Gumby/Fam105b gene cause an embryonic angiogenic phenotype in gumby mice. Gumby interacts with Disheveled 2 (Dvl2), is expressed in canonical Wnt-responsive endothelial cells and encodes an Ovarian Tumor Domain (OTU) class of deubiquitinase (DUB) that specifically cleaves linear ubiquitin linkages. A crystal structure of Gumby in complex with linear di-ubiquitin reveals how the identified mutations adversely impact substrate binding and catalytic function in line with the severity of their angiogenic phenotypes. Gumby interacts with HOIP/Rnf31, a key component of the linear ubiquitin assembly complex (LUBAC), decreases linear ubiquitination and activation of NFκB dependent transcription. This work provides support for the biological importance of linear (de)ubiquitination in angiogenesis, craniofacial and neural development and in modulating Wnt signaling.

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease (PLpro) has been implicated in playing important roles in virus maturation, dysregulation of host inflammation, and antiviral immune responses. The multiple functions of PLpro render it a promising drug target. Therefore, we screened a library of approved drugs and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising in vitro IC50 of 2.1 μM and an effective antiviral inhibition in cell-based assays. The co-crystal structure of SARS-CoV-2 PLproC111S in complex with GRL0617 indicates that GRL0617 is a non-covalent inhibitor and it resides in the ubiquitin-specific proteases (USP) domain of PLpro. NMR data indicate that GRL0617 blocks the binding of ISG15 C-terminus to PLpro. Using truncated ISG15 mutants, we show that the C-terminus of ISG15 plays a dominant role in binding PLpro. Structural analysis reveals that the ISG15 C-terminus binding pocket in PLpro contributes a disproportionately large portion of binding energy, thus this pocket is a hot spot for antiviral drug discovery targeting PLpro.

Varkud Satellite (VS) ribozyme mediates rolling circle replication of a plasmid found in the Neurospora mitochondria. We report crystal structures of this ribozyme at 3.1Å resolution, revealing an intertwined dimer formed by an exchange of substrate helices. Within each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional significance of active site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.

Summary RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by unusual 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here, we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small angle x-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the non-hydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain together with nucleotide binding, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.

Summary Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca2+-sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance (DEER) and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca2+. However, the favored orientation is one where the Ca2+-binding loops are oriented in opposite directions. A similar approach was taken for membrane associated C2A–C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca2+ and membrane binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca2+-dependent manner.

DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase ␣. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wildtype protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.DNA polymerase ␣-primase (pol-prim) 2 associates with eukaryotic replication forks in S-phase during the initiation of DNA replication (1, 2). pol-prim synthesizes a chimeric RNA-DNA primer of ϳ30 nucleotides that is then extended by more processive DNA polymerases that synthesize the leading and lagging strands. pol-prim is composed of four subunits (p180, p68, p58, and p48). The p180 subunit has the DNA polymerase catalytic activity and binds to both the p68 and p58 subunits. The p68 subunit has a regulatory function that is not completely understood. It is required for initiation of yeast chromosomal replication (3, 4) and cell-free SV40 DNA replication (5). In addition, phosphorylation of p68 alters the activity of polprim in SV40 replication (6 -9).The two smallest subunits, p48 and p58, together function as the DNA primase by creating an RNA primer of 7-10 nucleotides (10, 11). The p48 subunit contains the catalytic site (12). The p58 subunit stabilizes p48 and participates in initiation, elongation, and "counting" the ribonucleotides polymerized (13). Interestingly, p58 is also involved in transferring the RNA strand directly into the active site of the associated p180 subunit, which extends the growing nucleotide with dNTPs to complete the formation of the RNA-DNA primer (1, 14, 15). Knowledge of the molecular basis for regulation of the length of RNA portion of the primer an...