Structure of Fanzor2 reveals insights into the evolution of the TnpB superfamily

nature structural & molecular biology Brief Communication https://doi.org/10.1038/s41594-024-01394-4 Structure of Fanzor2 reveals insights into the evolution of the TnpB superfamily Received: 16 March 2024 Accepted: 27 August 2024 Published online: xx xx xxxx Check for updates Richard D. Schargel 1,3 , M. Zuhaib Qayyum Ravi C. Kalathur 2 & Elizabeth H. Kellogg 2 , Ajay Singh Tanwar2,3, 2,3 RNA-guided endonucleases, once thought to be exclusive to prokaryotes, have been recently identified in eukaryotes and are called Fanzors. They are classified into two clades, Fanzor1 and Fanzor2. Here we present the cryo-electron microscopy structure of Acanthamoeba polyphaga mimivirus Fanzor2, revealing its ωRNA architecture, active site and features involved in transposon-adjacent motif recognition. A comparison to Fanzor1 and TnpB structures highlights divergent evolutionary paths, advancing our understanding of RNA-guided endonucleases. Among the most widespread genes in all branches of life, transposons are potent agents of genetic change, as they mediate genome rearrangements through a variety of mechanisms. Recently, a group of transposon-encoded accessory proteins, termed obligate mobile element-guided activity (OMEGA) systems, were discovered to possess DNA cleavage activity, guided by a noncoding RNA called ωRNA, and are thought to be ancestors of Cas9 and Cas12 effectors from the class 2 clustered regularly interspaced short palindromic repeats (CRISPR) nuclease family1–3. One class of OMEGA proteins, called TnpB, has evolved into different Cas12 subtypes on multiple occasions, helping to explain the diversity of that family of CRISPR effectors4–7. Indeed, bioinformatics analyses of TnpB homologs indicate a wide range of both structure and function, with diverse protein architectures and catalytic-site geometries4,8. Eukaryotic TnpB homologs, referred to as Fanzors, can be identified in a wide range of organisms, including protists, fungi, arthropods, plants and eukaryotic viruses, and similarly function as RNA-guided nucleases6,7,9–11. Fanzors have attracted considerable interest as genome-editing tools, both for their natural functionality in eukaryotic cells and their substantially smaller size compared to Cas9 and Cas12 proteins. Fanzors are broadly categorized into two distinct clades, Fanzor1 and Fanzor2. Recent structural studies were conducted on Fanzor1 (refs. 9,10) but similar information remained lacking for the more compact Fanzor2. Here, we set out to fill that gap and characterize a representative member of the Fanzor2 clade to understand how these endonucleases recognize target DNA, as well as their relationship to Fanzor1 and the TnpB superfamily. Results Structure of ApmFz2–ωRNA–target DNA ternary complex We reconstituted a ternary complex of Acanthamoeba polyphaga mimivirus Fanzor2 (ApmFz2) with the native ωRNA scaffold and target DNA substrate11 and determined its structure. ApmFz2 constitutively associates with its ωRNA (247 nt); therefore, we coexpressed it with an ωRNA scaffold to promote complex stability. The ωRNA construct was designed with a hepatitis delta virus self-cleaving ribozyme at the 3′ end to produce a fixed-length 21-nt guide RNA (gRNA)12,13. The resulting ternary complex is biochemically active (Extended Data Fig. 1a) and exhibits cleavage activity consistent with previous results11. The target DNA substrate used in cryo-electron microscopy (cryo-EM) imaging was designed to form an RNA–DNA hybrid and promote ternary complex formation (Fig. 1a). The resulting 2.99 Å resolution cryo-EM map (Table 1 and Extended Data Fig. 1) enabled nearly complete building of both protein (468 of 520 aa) (Fig. 1b,c) and ωRNA (119 of 247 nt). The nuclear localization signal (NLS; corresponding to residues 1–53) is not observed, consistent with disorder predictions11. ApmFz2 is much more similar to TnpB than to a previously characterized Fanzor1 (ref. 9) (Extended Data Fig. 2), indicating a closer relationship to prokaryotic TnpB, at least at an architectural level, as previously suggested9–11. Like TnpB, ApmFz2 has a recognition (REC) domain, a wedge (WED) domain, a RuvC domain and a zinc finger (ZnF) domain (Fig. 1b). Notably, ApmFz2 has an N-terminal domain (NTD; residues 53–130) that is not observed in any other available TnpB structures (Extended Data Figs. 2 and 3). Part of this NTD (residues 65–91) appears to complete the RuvC fold Department of Microbiology, Cornell University, Ithaca, NY, USA. 2Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA. 3These authors contributed equally: Richard D. Schargel, M. Zuhaib Qayyum, Ajay Singh Tanwar. e-mail: ; 1 Nature Structural & Molecular Biology Brief Communication a https://doi.org/10.1038/s41594-024-01394-4 gRNA 226 nt Table 1 | Cryo-EM data collection, refinement and validation statistics Flexible 21 nt ApmFz2 ternary structure (EMD-44046), (PDB 9B0L) TS NTS Data collection and processing TAM b Magnification 1 131 143 254 309 320 NTD REC 53 65 1 NLS 470 500 520 WED 92 109 RuvC-R RuvC ZnF 200 Electron exposure (e per Å ) 51 Defocus range (μm) −0.5 to −2.5 Pixel size (Å) 1.044 Symmetry imposed C1 Initial particle images (no.) 1,801,494 Final particle images (no.) 189,912 Map resolution (Å) 2.99 FSC threshold 0.143 − 130 Thumb Not observed c 79,000 Voltage (kV) RuvC-R ωRNA RuvC ZnF 2 Map resolution range (Å) 2.28–5.83 Refinement 90° WED REC Initial model used (PDB code) None Model resolution (Å) 3.2 (unmasked) FSC threshold 0.5 Model resolution range (Å) Active site e d Guide Target DNA 90° Thumb Central channel 90° Map sharpening B factor (Å ) −86.4 Model composition 5 Nonhydrogen atoms 7,051 Protein residues 466 Ligands Mg: 1 Zn: 1 B factors (Å2) e d Not applicable 2 3’ WED Protein 108.32 Nucleotide 158.05 Ligand 148.23 Root-mean-square deviations REC TAM Glu467 Arg500 Asp501 Thumb Asp324 ZnF 0.004 Bond angles (°) 0.582 Validation RuvC Fig. 1 | Cryo-EM structure of ApmFz2 ternary complex. a, Diagram showing target DNA substrate (bottom) annealed to ωRNA (top). Nucleotides not observed in the cryo-EM structure are light gray. The gRNA is pink and the TAM is purple. On the DNA molecule, TS marks the target strand and NTS marks the nontarget strand. b, Top: domain organization of ApmFz2, with domain boundaries indicated by residue numbers. NTD, white; REC, aqua; WED, orange-yellow; RuvC, green; ZnF, pink. Bottom: detailed annotation of the NTD. The NLS was not observed in the cryo-EM map. The RuvC-R (blue-green) and thumb (blue) regions structurally reinforce the RuvC and REC domains, respectively. Gray boxes indicate linker regions not specifically assigned to a domain. c, Cryo-EM reconstruction (top) and atomic model (bottom) of ApmFz2 ternary complex. Domains and nucleic acid molecules are the same colors as in a,b, except for ωRNA, which is shown i (...truncated)