Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

Abstract

CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB’s tetrameric architecture is stabilized by a different set of protein–protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5′ nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor–DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue “hook” that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.

Structures of the holo CRISPR RNA-guided transposon integration complex

“…To test whether domain IIΔ is generally required, we next tested various TnsB fragments for their ability to stimulate ATP hydrolysis. TnsB C-terminal constructs (called CTD, residues 476-584) and TnsB C-terminal truncations (ΔCTD, residues 1-475) both fail to stimulate ATP hydrolysis, as expected based on our previous work ( 18 ). In contrast, C-terminal constructs including domain IIΔ (IIΔ+CTD, residues 410-584) increased ATP-hydrolysis levels over basal levels of TnsC ATPase activity (i.e.…”

“…TnsB is a RNaseH transposase that bears significant similarities to MuA from bacteriophage Mu ( 18, 22 ). TnsB contains multiple functionally distinct domains dedicated to DNA-binding, catalytic activity, or interactions with the AAA+ regulator TnsC ( 18 ) ( Fig. S8 ).…”

“…S8 ). TnsB Hook serves as the point of contact with the TnsC filament body and is critically important for CAST transposition ( 18 ). We identified well-defined density corresponding to the TnsB ’Hook’ (TnsB Hook ,) at four of the six TnsC protomers closest to TnsB (TnsC protomers 2-5, Fig.…”

“…In the ShCAST system, the transposase TnsB is recruited to the target site via TnsC ( Fig. 1A, left ) ( 18 ). This is intrinsically tied to ATP-hydrolysis, because substitution with a non-hydrolyzable ATP analog (AMP-PNP) inhibits TnsB-mediated filament disassembly and results in non-targeted transposition ( 19 ).…”

“…Therefore, a crucial role of TnsB is also to stimulate TnsC’s ATP-hydrolysis activity in the process of target site selection. Two TnsB-TnsC interactions have been biochemically demonstrated to be required for this process, one of which corresponds to TnsB Hook and the other one located elsewhere in full-length TnsB ( 18 ). Excitingly, within the transpososome structure we observe a second TnsB-TnsC interaction, not previously observed, at the C-terminal face of TnsC ( Fig.…”