Crystal Structure of I-Dmoi in Complex with Its Target DNA Provides New Insights Into Meganuclease Engineering

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering María Jose´ Marcaidaa,1, Jesu´ s Prietob,1, Pilar Redondoa, Alejandro D. Nadrac, Andreu Alibe´ sc, Luis Serranoc,d, Sylvestre Grizote, Philippe Duchateaue, Fre´ de´ ric Paˆ quese, Francisco J. Blancob,f, and Guillermo Montoyaa,2 aMacromolecular Crystallography and bNuclear Magnetic Resonance Groups, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre, c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain; cEuropean Molecular Biology Laboratory-Centre for Genomic Regulation Systems Biology Unit, Centre de Regulacio´Geno`mica, Parc de Recerca Biome`dica de Barcelona-Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain; dInstitucioĆatalana de Recerca I Estudis Avançats and fStructural Biology Unit, Centro de Investigacioń Cooperativa bioGUNE, Parque Tecnolo´gico de Vizcaya, Edificio800, 48160 Derio, Spain; and eCellectis S.A., 102 Route de Noisy 93235 Romainville, France Edited by Marlene Belfort, New York State Department of Health, Albany, NY, and approved September 24, 2008 (received for review May 17, 2008) Homing endonucleases, also known as meganucleases, are sequence- or intermonomer interface. The last acidic residue of this LAGLI- specific enzymes with large DNA recognition sites. These enzymes DADG motif participates in DNA cleavage by a metal ion depen- can be used to induce efficient homologous gene targeting in cells dent mechanism of phosphodiester hydrolysis (4). and plants, opening perspectives for genome engineering with A combinatorial and rational approach (10) has been used for applications in a wide series of fields, ranging from biotechnology engineering the overall specificity of the homodimeric meganucle- to gene therapy. Here, we report the crystal structures at 2.0 and ase I-CreI to cleave specific sequences, such as the human RAG1 2.1 Å resolution of the I-DmoI meganuclease in complex with its and XPC genes (11, 12). However, the resulting variants are substrate DNA before and after cleavage, providing snapshots of heterodimers, consisting of 2 engineered monomers that need to be the catalytic process. Our study suggests that I-DmoI requires only coexpressed in the targeted cell. As a consequence, 2 undesired 2 cations instead of 3 for DNA cleavage. The structure sheds light homodimers could be produced in addition to the heterodimer, onto the basis of DNA binding, indicating key residues responsible contributing to a loss of efficiency and specificity. This problem for nonpalindromic target DNA recognition. In silico and in vivo could be solved by strategies such as dimer engineering (13) or analysis of the I-DmoI DNA cleavage specificity suggests that considering monomeric meganuclases. despite the relatively few protein-base contacts, I-DmoI is highly I-DmoI is a monomeric meganuclease from the hyperthermo- specific when compared with other meganucleases. Our data open philic archaeon Desulfurococcus mobilis; its structure in the absence the door toward the generation of custom endonucleases for tar- of bound DNA has been solved (7). However, there was scant geted genome engineering using the monomeric I-DmoI scaffold. information at the molecular level about I-DmoI DNA recognition and cleavage mechanisms because no structure of the enzyme- gene targeting ͉ genetics ͉ protein–DNA interactions ͉ DNA complex was available. The absence of these data has X-ray crystallography hampered the use of I-DmoI as a scaffold to engineer tailored specificities. The crystal structures of I-DmoI together with the results of in eganucleases are sequence-specific enzymes that recognize silico predictions and in vivo experiments open new possibilities to Mlarge (12–45 bp) DNA target sites. These enzymes are often engineer custom specificities, using I-DmoI as scaffold. encoded by introns or inteins behaving as mobile genetic elements. Results and Discussion They produce double strand breaks (DSB) that get repaired by homologous recombination with an intron- or intein-containing Overall Structure of the I-DmoI/DNA Complex. I-DmoI in complex gene, resulting in the insertion of the intron or intein in that with a 25-bp DNA was crystallized as an enzyme-substrate complex 2ϩ 2ϩ particular site (1). Meganucleases are being used to stimulate with Ca and as an enzyme-product complex with Mn . The homologous gene targeting in the vicinity of their target sequences overall fold of I-DmoI in complex with its DNA target (Fig. 1) with the aim of improving current genome engineering approaches, shows a clear pseudo 2-fold symmetry axis between the 2 LAGLI- ␣ ␣ alleviating the risks due to the randomly inserted transgenes (2, 3). DADG helices ( 1 and 4) dividing the protein into 2 domains, A The use of meganuclease-induced recombination has long been (residues 5–98) and B (residues 103–195). These domains contain ␣␤␤␣␤␤␣ limited by the repertoire of natural meganucleases. In nature, the typical topology of the LAGLIDADG family, al- ␤ meganucleases are essentially represented by homing endonucle- though domain B contains only 3 -strands. Two antiparallel ␤ ␤ ␤ ases, a family of enzymes encoded by mobile genetic elements -sheets, composed of strands 1–4 in domain A and 5–7 in whose function is to initiate DSB induced recombination events in domain B, form a concave surface with an inner cylindrical shape a process referred to as homing (4). The probability of finding a homing endonuclease cleavage site in a chosen gene is extremely Author contributions: M.J.M., J.P., P.D., F.P., F.J.B., and G.M. designed research; M.J.M., J.P., low. Thus, making artificial meganucleases with custom-made P.R., A.D.N., A.A., S.G., P.D., F.J.B., and G.M. performed research; F.J.B. contributed new substrate specificity is an intense area of research. reagents/analytic tools; M.J.M., J.P., P.R., A.D.N., A.A., L.S., S.G., P.D., F.P., F.J.B., and G.M. Sequence homology has been used to classify homing endonucle- analyzed data; and M.J.M., J.P., L.S., F.J.B., and G.M. wrote the paper. ases into 5 families, the largest one having the conserved LAGLI- The authors declare no conflict of interest. DADG sequence motif. Homing endonucleases containing one This article is a PNAS Direct Submission. such motif function as homodimers. In contrast, homing endo- Data deposition: The atomic coordinates have been deposited in the Protein Data Base, nucleases containing 2 motifs are single chain proteins (5, 6). www.pdb.org (PDB ID codes 2VS7 and 2VS8). Structural information for several members of the LAGLIDADG 1M.J.M. and J.P. contributed equally to this manuscript. endonuclease family indicate that these proteins adopt a similar 2To whom correspondence should be addressed. E-mail: [email protected]. active conformation as homodimers or as monomers with 2 sepa- This article contains supporting information online at www.pnas.org/cgi/content/full/ rate domains (7–9). The LAGLIDADG motifs form structurally 0804795105/DCSupplemental. conserved ␣-helices tightly packed at the center of the interdomain © 2008 by The National Academy of Sciences of the USA 16888–16893 ͉ PNAS ͉ November 4, 2008 ͉ vol. 105 ͉ no. 44 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804795105 Downloaded by guest on October 1, 2021 Fig. 1. Crystal structure of I-DmoI in complex with its target DNA. (A) The protein moiety is colored accord- ing to its secondary structure (␣-helices are in blue, ␤-strands are in orange, and loops are in gray), the complex is shown in 2 different orientations. The cal- cium ion is shown as a green sphere. (B) Oligonucleo- tide used for crystallization. Throughout the text the individual bases are named with a subindex strandA or strandB indicating the DNA strand they belong to. where the DNA molecule is accommodated. The substrate and ions in the active site has been reported to be 3 or 2 (Fig. S4) product bound structures differ in the DNA molecule, with both (17–19), only 1 Ca2ϩ anomalous peak could be detected in the strands cleaved at the expected sites in the Mn2ϩ bound complex I-DmoI active center (Fig. 2A). The Ca2ϩ atom is hexa-coordinated (Figs. 1B and 2) (14). However, the protein moieties are very with phosphates from both DNA strands (2AstrandA and similar, C␣1–179 rmsd 0.21 Å. Ϫ3CstrandB), 3 water molecules and D21 from the first LAGLI- The structure of the ␣-helical core of the protein scaffold is DADG motif. The additional anomalous difference study in the similar to the I-DmoI structure in the absence of the DNA duplex presence of Mn2ϩ demonstrated binding of 2 metal ions with (7). However, there are differences in the overall conformation of equivalent occupancy, similarly to the Mg2ϩ positions reported for the ␤-strands, the loops that join them and the connections with the I-AniI (20) (Fig. 2B and Fig. S4). In contrast, 3 Mg2ϩ were reported core helices, that favor embracing the DNA molecule (supporting for I-CreI (17) and H-DreI active sites (15), even though in the last information (SI) Fig. S1). All these changes are reflected in a case the DNA remained intact. Two of the metal ions were C␣1–179 rmsd of 1.27 Å between the I-DmoI and I-DmoI/DNA unshared, whereas the central one was shared between the 2 complex structure. The structure of the I-DmoI/DNA complex also catalytic residues (Fig. S4). In I-DmoI, the central site does not show depicts differences when compared with H-DreI (Fig. S2) (15), a any anomalous signal and therefore the density is modeled as a chimerical enzyme that contains the domain A of I-DmoI fused to an water molecule. Both I-DmoI domains cooperate in metal ion I-CreI monomer (originally named E-DreI).

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