Graphical Abstract Highlights d Crystal structures of Cas9-bound AcrIICs suggest distinct inhibitory mechanisms d AcrIIC2 interferes with RNA-and DNA-loading steps through binding to Cas9 BH motif d AcrIIC3 induces Cas9 dimerization by interacting with the HNH domain and REC lobe d Cas9 enzymes can be reengineered to become susceptible to AcrIIC's inhibition

Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used alone for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by an immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLNs. A low dose of I-R-F induces not only high titers of long-lasting neutralizing antibodies (NAbs) but also more comprehensive T cell responses than RBD. Notably, I-R-F provides comprehensive protection in the form of a one-dose vaccine without an adjuvant. Our study shows that the pan-epitope modified human I-R-F (I-P-R-F) vaccine provides rapid and complete protection throughout the upper and lower respiratory tracts against a high-dose SARS-CoV-2 challenge in rhesus macaques. Based on these promising results, we have initiated a randomized, placebo-controlled, phase I/II trial of the human I-P-R-F vaccine (V-01) in 180 healthy adults, and the vaccine appears safe and elicits strong antiviral immune responses. Due to its potency and safety, this engineered vaccine may become a next-generation vaccine candidate in the global effort to overcome COVID-19.