Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation

Abstract

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase-loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3'-phosphotyrosine protein-DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre-loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a 'cleavage-competent' Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3'-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the 'cleavage-competent' subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the 'non-cleaving' subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.

Figure 1

(A) Schematic representation of Cre–loxP-mediated recombin ation. Cre molecules are represented by ellipses (green for non-cleaving subunits and orange for active subunits) and labeled A, B, A′ and B′. Red and black spheres indicate cleavage sites. This figure is adapted from Gopaul et al. (8). (B) Sequences of the loxP (used in this study) and the symmetrized loxS (used by Van Duyne’s group) DNA. The 13 bp inverted repeat sequences are indicated with black arrows. The thymidine position substituted by a 5-iodo-deoxyuridine is highlighted. The spacer region is boxed and arrowheads depict cleavage sites. Residues noted in lower case were added at the 3′ and 5′ ends for crystallization of the complex. (C) In the Cre–loxP synaptic complex, the 8 bp spacer region is kinked immediately next to the top strand cleavage site (top). After the first cleavage, a 3′-phosphotyrosine covalent intermediate is formed (middle). Some rearrangements in the spacer region redistribute the kink over several residues in the cleaved DNA but not all base pairs are still formed. Unlike in the present structures, in the Cre–loxS synaptic complex, the kink is 5 bp away from the cleavage site (bottom).

Figure 2

In vitro recombination experiments performed on native loxP sites or modified by the introduction of a phosphorothioate at the protein cleavage site. Lanes marked ‘48’ contain the 48mer loxP oligomer. Lanes containing the recombinase are indicated with ‘Cre’. The 37mer loxP oligomer is present in lanes marked with ‘5/9’ (unmodified DNA), ‘2/9’ (phosphorothioate on the top strand), ‘5/6’ (phosphorothioate on the bottom strand) and ‘2/6’ (phosphorothioate on top and bottom strands). Due to the presence of overhangs on the 37mer loxP site used for crystallization experiments, two different sizes of recombinant DNA are obtained with unmodified loxP site, corresponding to a 42mer and a 43mer oligomers. No recombinant DNA was detectable in our conditions when a phosphorothioate was present either on the top or the bottom strand.

Figure 3

(A) View of the synaptic tetramer as observed in the asymmetric unit of the present structure. The two DNA duplexes are in black and red. The four Cre proteins subunits are in dark green, light green (non-cleaving subunits), orange and red (cleaving subunits). (B) View of the 3F_obs–2F_calc electron density map (contoured at 1.4 σ level) around A-T and G-C base pairs showing the overall quality of the electron density map. Note the additional density observed for N2 of guanines compared with adenines and C5 of thymines compared with cytidines.

Figure 4

(A) Stereoview of the 3F_obs–2F_calc electron density map (contoured at 1.4 σ level) around the active site of the ‘cleavage-competent’ Cre subunit and the kink region of the loxP site. A metal ion (possibly a magnesium) is indicated by an orange sphere. (B) DNA backbone superposition of the wild-type loxP sequence as observed in the present structure (in blue) and the symmetrized loxS sequence (PDBID 4CRX; the second duplex was generated by applying 2-fold symmetry). While the overall bending is the same, marked differences are found in the spacer region resulting from different locations of kinks. (C) Stereoview of the 3F_obs–2F_calc electron density map (contoured at 1.4 σ level) showing a magnesium-mediated protein–DNA contact.

Figure 5

(A) Stereoview of a Cre dimer (represented by the solvent accessible surface of the protein) interacting with a loxP site (the spacer region is in red). The ‘cleavage-competent’ Cre (in orange) is bound close to the top strand cleavage site (ApT step). A close-up view of the spacer region shows that a sharp kink (circled in pink) is located at the TG/CA step next to the scissile phosphate in the top strand. The scissile bond is represented by a red sphere and indicated by a red arrowhead. (B) Schematic drawing of protein–DNA contacts in the spacer region. Amino acids are colored in green or orange depending on whether they belong to the ‘non-cleaving’ or the ‘cleavage-competent’ subunit, respectively. Note that except for the scissile phosphate, very few contacts are formed with the top strand, facilitating its exchange after cleavage. Nucleotide numbering follows the one used in PDB entries. Top and bottom strands are depicted in dark and light gray, respectively. The scissile phosphate of the top strand is indicated as a red sphere.

Figure 6

Stereoview of the active site of Cre recombinase in the Cre–loxP complex. (A) In the cleavage-competent subunit of the pre-cleavage Cre–loxP synaptic complex, (B) the cleavage-competent subunit of the 3′-phosphotyrosine covalent intermediate and (C) in the non-cleaving subunit. The 3F_obs–2F_calc electron density map contoured at 1.4 σ level is in blue. The scissile phosphate is represented in yellow.

Figure 7

Stereoviews showing a superposition of the active site of several Cre–DNA complexes for the ‘cleavage-competent’ (top) and ‘non-cleaving’ (bottom) subunits. Proteins are colored as follows: purple, Cre–loxS synaptic complex (4CRX); light green, Cre-immobile HJ intermediate complex (2CRX); dark green, Cre-wild-type HJ intermediate complex (1KBU); blue, Cre–loxA suicide substrate (3′-phosphotyrosine covalent intermediate); yellow, Cre–LoxP synaptic complex (this study, 1nzb); orange, Cre–loxP 3′-phosphotyrosine covalent intermediate (this study, 1ouq).

Figure 8

Stereoviews of a Cre dimer showing inter-subunits contacts in the neighborhood of the active site. The non-cleaving subunit is in green and its loop 198–208 in purple. Cleavage-competent subunits before cleavage (in yellow) and in the 3′-phosphotyrosine covalent intermediate (in orange) are superimposed. The moving C-terminal part containing helices M and N is in red. The close-up view (top) is from a different point of view to highlight the movement of helices M and N.