A subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site. This bound structure potentially competes with a stable but non-productive RNA structure. At the cleavage site, MmCas6b supplies a base pair mimic to stabilize a short 2 base pair stem immediately upstream of the scissile phosphate. Complementary biochemical analyses support the dual-AAYAA binding model and a critical role of the protein-RNA base pair mimic. Our results reveal a previously unknown method of processing non-stem-loop CRISPR RNA by Cas6.

About one-third of the more than 300 C2H2 zinc finger proteins of Drosophila contain a conserved sequence motif, the zinc finger-associated domain (ZAD). Genes that encode ZAD proteins are specific for and expanded in the genomes of insects. Only three ZAD-encoding gene functions are established, and the role of ZAD is unknown. Here we present the crystal structure of the ZAD of Grauzone (ZAD(Grau)), a Drosophila transcription factor that specifically controls the maternal Cdc20-like APC subunit Cortex. ZAD forms an atypical treble-clef-like zinc-coordinating fold. Head-to-tail arrangement of two ZAD(Grau) molecules in the crystals suggests dimer formation, an observation supported by crosslinking and dynamic light scattering. The results indicate that ZAD provides a novel protein-protein interaction module that characterizes a large family of insect transcription factors.

Ubiquitin-conjugating enzymes (E2s) govern key aspects of ubiquitin signaling. Emerging evidence suggests that the activities of E2s are modulated by posttranslational modifications; the structural underpinnings, however, are largely unclear. Here, we unravel the structural basis and mechanistic consequences of a conserved autoubiquitination event near the catalytic center of E2s, using the human anaphase-promoting complex/cyclosome-associated UBE2S as a model system. Crystal structures we determined of the catalytic ubiquitin carrier protein domain combined with MD simulations reveal that the active-site region is malleable, which permits an adjacent ubiquitin acceptor site, Lys+5, to be ubiquitinated intramolecularly. We demonstrate by NMR that the Lys+5-linked ubiquitin inhibits UBE2S by obstructing its reloading with ubiquitin. By immunoprecipitation, quantitative mass spectrometry, and siRNA-and-rescue experiments we show that Lys+5 ubiquitination of UBE2S decreases during mitotic exit but does not influence proteasomal turnover of this E2. These findings suggest that UBE2S activity underlies inherent regulation during the cell cycle.

Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.