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Crystal structure of the V(D)J recombinase RAG1–RAG2

Nature volume 518, pages 507–511 (2015)Cite this article

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Abstract

V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1–RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1–RAG2 complex at 3.2 Å resolution. The 230-kilodalton RAG1–RAG2 heterotetramer is ‘Y-shaped’, with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1–RAG2 heterodimer composes one arm of the ‘Y’, with the active site in the middle and RAG2 at its tip. The RAG1–RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.

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Figure 1: Structure determination of RAG1–RAG2 recombinase.
Figure 2: Crystal structure of RAG1–RAG2.
Figure 3: The RAG1 structure.
Figure 4: The interface between RAG1 and RAG2.
Figure 5: A RAG1–RAG2–DNA model.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession code 4WWX.

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Acknowledgements

We thank G. Grundy and S. Ramon-Maiques for the pioneering work that made this study possible, and D. Leahy for editing the manuscript. The research was supported by the intramural research program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

Author information

Author notes

  1. Mikalai Lapkouski

    Present address: Present address: Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden, and Centre for Structural Systems Biology, DESY, 22607 Hamburg, Germany.,

Authors and Affiliations

  1. Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, Maryland 20892, USA,

    Min-Sung Kim, Mikalai Lapkouski, Wei Yang & Martin Gellert

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Contributions

M.-S.K. prepared SEC complexes and carried out crystallography. M.L. developed protocols for protein expression and initial crystallization. W.Y. and M.G. designed the project, and M.-S.K., W.Y. and M.G. prepared the manuscript.

Corresponding authors

Correspondence to Wei Yang or Martin Gellert.

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Extended data figures and tables

Extended Data Figure 1 Structure determination.

a, The plot of correlation coefficient (CC) of anomalous signal versus resolution. The red line indicates the cutoff of CC = 0.3. Merging data from the two best crystals produced a better CC than merging data from all six crystals. The data processing procedure is outlined above the plot20. b, The SAD experimental map contoured at 1.3σ showed the content of an asymmetric unit. The Se anomalous map is contoured at 3.0σ in red. c, A typical crystal of RAG1–RAG2. d, The content of crystals was examined by protein and DNA denaturing gels after a thorough wash of the crystals and stained by Coomassie blue and SYBR green. To confirm the 1:1 molar ratio of 12 and 23RSS DNA, 32P-labelled input RSS DNAs and those in SEC complexes before and after crystallization are shown beneath the SYBR-green-stained DNA gel. e, Transposition assay of the purified SEC (RAG1–RAG2–12/23RSS DNA complex) used for crystallization. Supercoiled pUC19 (sc, with a small amount of open circle, oc) was the target; it was linearized by HindIII as a control. The SEC (0.25, 0.5 and 1.0 μM) was active in concerted transposition and thus linearizing pUC19. In contrast, RAG1–RAG2 or HMGB1 (0.5 µM) each alone was not active. f, Crystal packing of neighbouring RAG1–RAG2 complexes (shown in dark and light colours) occludes one nonamer-binding site in each heterotetramer of RAG1–RAG2.

Extended Data Figure 2 RAG2 core fragment (1–351 amino acids) is active.

a, Sequence alignment of RAG2 from mouse (320–387 amino acids), human, rat and Xenopus with predicted secondary structures shown above. b, Core RAG2 (1–387) and two further truncated RAG2 variants (1–351 and 1–367) were constructed with a non-cleavable N-terminal MBP tag and co-expressed with the tag-less core RAG1. The Coomassie blue R-250 stained SDS gel shows the purified RAG1–RAG2 complexes. c, Purified RAG1–RAG2 complexes with truncated RAG2 variants are equally active in cleaving a 32P-labelled 12RSS DNA (in the presence of a 23RSS and Mg2+, as examined by TBE-Urea gel). d, Elution profiles of RAG1–RAG2 (both long and short forms) complexed with DNA from Superdex-200 (S200) in a low salt buffer (50 mM HEPES pH 7.0, 60 mM KCl, 1 mM maltose and 2 mM DTT). Regardless of the length of RAG2, the major S200 eluant peak came out at the same time point and contained RAG1, RAG2 (1–351 or 1–387) and HMGB1 proteins, as shown in the SDS gel (right insert), as well as 12 and 23RSS oligonucleotides, as confirmed by a TBE-Urea gel stained by SYBR green (left insert).

Extended Data Figure 3 Comparison of RAG2 with β-propeller and β-pinwheel structures.

KLHL2 (PDB 4CHB)56 is selected to represent the β-propeller proteins, and the C-terminal domain (CTD) of GyrA (PDB 1SUU)57 is selected to represent the β-pinwheel structures. After superposition, RAG2 (a), KLHL2 (b) and GyrA (c) are shown side-by-side individually in two orthogonal views. Each structure is coloured from N to C terminus in blue to red rainbow colours. The loops in RAG2 that interact with RAG1 are labelled. The six β-blades are named by Roman numerals, I–VI, from N to C terminus; four β-strands in each blade are named by Arabic numerals, 1–4.

Extended Data Figure 4 Comparison of RAG1 and NBD–DNA complex.

a, The NBD in the RAG1–RAG2 core complex (blue and green) superimposes well with the published structure of the NBD–DNA complex (PDB 3GNA, protein coloured yellow)22. b, The twelve SCID/Omenn syndrome mutations in the NBD domain are mapped onto the crystal structure of the NBD–DNA complex. Six SCID/Omenn syndrome (R391 to R407) mutations are located on a positively charged surface patch that interacts with the nonamer; five remaining SCID/Omenn syndrome mutations (L408 to A441) appear to affect the structural integrity of the NBD, and R446 may interact with the spacer DNA in each RSS.

Extended Data Figure 5 Transposases that form a hairpin intermediate.

a–c, Hermes (PDB 4D1Q)37 (a), bacterial Tn5 (PDB code 1MUS)40 (b), and RAG1 dimers (c) are shown as ribbon diagrams in two orthogonal views, with the dyad perpendicular to the viewing plane (left) or in the plane (right). Each dimer consists of a cyan and a green subunit. The catalytic RNH domains are highlighted in pink, and the conserved catalytic residues are shown as red ball-and-sticks. The catalytic divalent metal ions are shown as green spheres if present. The DNAs, coloured in yellow (cleaved by the cyan subunit) and orange (cleaved by the green subunit), have similar orientations in the Hermes and Tn5 complexes (as indicated by the arrows). Arrows with dashed outlines indicate that the DNAs are in the back of the viewing plane. Notably, the pair of RNH domains is oriented similarly in all three cases. The predicted orientations of DNAs bound to RAG1 are indicated by the yellow and orange arrows, and the α-helices connected to the third catalytic carboxylates (shown in light purple) probably bridge two DNAs in RAG1 recombinase as in Hermes and Tn5.

Extended Data Figure 6 Transposases that do not form a hairpin intermediate.

a–c, Retroviral integrase from Prototype foamy virus (Pfv, PDB 3OS0)36 (a), bacterial MuA transposase (PDB code 4FCY)38 (b) and eukaryotic Mos1 mariner transposase (PDB 3HOT)39 (c) are shown in comparable views and same representations as Hermes, Tn5 and RAG1–RAG2 in Extended Data Fig. 5. Each catalytic dimer consists of a cyan and a green subunit. Two accessory subunits in Pfv are shown in light blue and green, and two accessory subunits of the MuA structure are omitted for clarity. The catalytic RNH domains are highlighted in pink. The DNAs, coloured in yellow (cleaved by the cyan subunit) and orange (cleaved by the green subunit), have similar orientations (within 30°) as indicated by the arrowheads, but each differs more than 90° from the corresponding DNA in Hermes or Tn5 transposase. The grey DNA in the MuA complex represents the target of transposition. Among these three recombinases, the α-helix that follows the third catalytic carboxylate (coloured in light purple) does not cross over to interact with a second DNA.

Extended Data Figure 7 Surface potential and conservation of RAG1–RAG2 complex.

a, Orthogonal views of the electrostatic potential surface of the RAG1–RAG2 structure. Blue indicates positive charges, and red negative. b, Orthogonal views of the molecular surface of RAG1–RAG2 with absolutely conserved residues highlighted in deep purple. The NBD is well conserved. The views with dyad in the plane here are related to the image shown in Fig. 5c by ∼50° rotation around the dyad.

Extended Data Table 1 Statistics of native and SeMet SAD data collection and structure refinement
Full size table
Extended Data Table 2 Missense mutations of RAG1 and RAG2 identified in human SCID/OS patients
Full size table
Extended Data Table 3 Mouse RAG1–RAG2 mutations presented in ref. 4
Full size table

Supplementary information

Animation of the SEC model

RAG1/2 is shown in surface representation. Blue and red represent positively and negatively charged surface potentials. The nonamers are modeled after the NBD-DNA complex structure (12bp, PDB: 3GNA), after domain superposition. Each 16bp DNA including the heptamer mimic is brought in by superposition of the Hermes-DNA complex (PDB: 4D1Q) with the RNH domain of each RAG1 subunit. The view shows the 12RSS-like DNA, where the 16bp and 12bp DNAs are nearly touching. The 23RSS-like DNA is in the back, and the two DNAs modeled are ~30Å apart. (MOV 4452 kb)

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Kim, MS., Lapkouski, M., Yang, W. et al. Crystal structure of the V(D)J recombinase RAG1–RAG2. Nature 518, 507–511 (2015). https://doi.org/10.1038/nature14174

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