Structural plasticity enables broad cAn binding and dual activation of CRISPR-associated ribonuclease Cdn1

TL;DR

A CRISPR accessory protein called Cdn1 can bind and be activated by multiple cyclic oligoadenylates, showing structural flexibility that enhances microbial defense against viruses.

Contribution

Discovery of a CRISPR-associated protein with broad cyclic oligoadenylate binding and dual activation through structural plasticity.

Findings

Cdn1 binds to cA3, cA4, and cA6 but is activated by cA4 and cA6 with varying efficacy.

Structural analysis reveals conformational changes upon cyclic oligoadenylate binding, enabling RNA cleavage.

The dual activation mechanism reflects evolutionary adaptation for enhanced antiviral defense.

Abstract

Prokaryotes have naturally evolved diverse RNA-guided defense systems against viral infections, with the type III CRISPR–Cas systems representing the most intricate. These systems feature accessory proteins activated by cyclic oligoadenylates (cOAs) produced upon target RNA recognition, synergizing with the CRISPR–Cas machinery to defend against exogenous invaders. Typically, each accessory protein is activated by only one specific cOA type. Here, we characterize Cdn1, a type III-B CRISPR accessory protein from Psychrobacter lutiphocae, which binds to cA3, cA4, and cA6, but activated by cA4 and cA6 with different efficacies to catalyze ssRNA cleavage. Combined structural and biochemical analyses reveal that cOA binding triggers dramatic conformational reorganization, including the formation of a dimerization interface of nuclease domains, the emergence of substrate binding cleft, and the reconstruction of a metal-dependent catalytic center essential for RNA cleavage. This dual activation mechanism illustrates evolutionary innovation within CRISPR-associated Rossman-fold nucleases. We propose that such structural plasticity evolved to maximize defensive resilience during microbial competition and horizontal gene transfer, while preserving broad-spectrum antiviral ability. These findings not only elucidate the activation mechanisms of Cdn1 within the type III systems but also underscore the functional complexity and adaptability of CRISPR–Cas ancillary proteins.

Graphical Abstract

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