Target RNA-guided protease activity in type III-E CRISPR-Cas system.

The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.

Molecular and structural basis of an ATPase-nuclease dual-enzyme anti-phage defense complex.

Coupling distinct enzymatic effectors emerges as an efficient strategy for defense against phage infection in bacterial immune responses, such as the widely studied nuclease and cyclase activities in the type III CRISPR-Cas system. However, concerted enzymatic activities in other bacterial defense systems are poorly understood. Here, we biochemically and structurally characterize a two-component defense system DUF4297-HerA, demonstrating that DUF4297-HerA confers resistance against phage infection by cooperatively cleaving dsDNA and hydrolyzing ATP. DUF4297 alone forms a dimer, and HerA alone exists as a nonplanar split spiral hexamer, both of which exhibit extremely low enzymatic activity. Interestingly, DUF4297 and HerA assemble into an approximately 1 MDa supramolecular complex, where two layers of DUF4297 (6 DUF4297 molecules per layer) linked via inter-layer dimerization of neighboring DUF4297 molecules are stacked on top of the HerA hexamer. Importantly, the complex assembly promotes dimerization of DUF4297 molecules in the upper layer and enables a transition of HerA from a nonplanar hexamer to a planar hexamer, thus activating their respective enzymatic activities to abrogate phage infection. Together, our findings not only characterize a novel dual-enzyme anti-phage defense system, but also reveal a unique activation mechanism by cooperative complex assembly in bacterial immunity.

Cryo-EM structures of African swine fever virus topoisomerase.

IMPORTANCE: African swine fever virus (ASFV) is a highly contagious virus that causes lethal hemorrhagic diseases known as African swine fever (ASF) with a case fatality rate of 100%. There is an urgent need to develop anti-ASFV drugs. We determine the first high-resolution structures of viral topoisomerase ASFV P1192R in both the closed and open C-gate forms. P1192R shows a similar overall architecture with eukaryotic and prokaryotic type II topoisomerases, which have been successful targets of many antimicrobials and anticancer drugs, with the most similarity to yeast topo II. P1192R also exhibits differences in the details of active site configuration, which are important to enzyme activity. These two structures offer useful structural information for antiviral drug design and provide structural evidence to support that eukaryotic type IIA topoisomerase likely originated from horizontal gene transfer from the virus.

Structure and function of a bacterial type III-E CRISPR-Cas7-11 complex.

The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR-Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA-target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.