Structural insight into IscB's RNA-lid-based inactivation mechanism.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• IscB, a compact Cas9 ancestor, shows promise as a programmable genome editor due to its small size and therapeutic delivery potential.
• Limited structural insights exist for IscB regulation, with only a target-bound R-loop structure previously reported.

Purpose of the Study:

• To elucidate the structural trajectory of an engineered IscB from a resting state to activation.
• To uncover the molecular mechanisms of IscB autoinhibition and activation.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to resolve four high-resolution structures of IscB.
• Structures captured include the apo resting state, intermediate complexes with varying guide-target pairing (6-nt and 10-nt), and a fully paired 16-nt primed cleavage state.

Main Results:

• A dual inactivation mechanism mediated by RNA lids (ωRNA and guide RNA lids) was identified, blocking HNH and RuvC active sites respectively.
• Guide RNA undergoes stepwise displacement ('car pedal' motion) triggering activation at 11-nt pairing.
• The HNH domain stabilizes the R-loop via an R-wedge motif and undergoes activation-driven rotation.
• Engineering hinge motifs in IscB variants (IscBHig1, IscBHig2) enhanced conformational flexibility and genome-editing efficiency in cells.

Conclusions:

• The study reveals the molecular basis of IscB autoinhibition and activation.
• Previously uncharacterized regulatory features of IscB were identified.
• Hinge elements are established as a target for engineering compact and efficient genome editors.