期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：32
DOI：10.1073/pnas.2202590119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
CRISPR-associated transposons (CASTs) show tremendous promise for genome engineering yet remain poorly understood. Here, we present the cryo-electron microscopy structure of the transposase (TnsB) from the V-K CAST element from
Scytonema hofmanni (ShCAST). We determine the molecular mechanism of TnsB recruitment to the target site (via the AAA+ regulator TnsC) and the structural details of the TnsB transposase. This TnsB structure reveals architectural similarities to MuA, but also key structural differences that are significant for understanding CAST transposition. Importantly, we highlight a base-flipping mechanism for stabilizing the 5′ end of the transposon, potentially to ensure the fidelity of synaptic complex assembly. The structures presented here provide a direct target for rational, structure-guided design strategies and re-engineering of CAST elements.
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.
