Nucleic Acids Research, 2008, Vol. 36, No. 19 6115 Figure 5. Flipped base binding pockets of (A and B) PspGI and (C and D) Ecl18kI. In case of PspGI (A and B), only residues closer than 4 A˚ to the ﬂipped nucleosides are shown. In the case of Ecl18kI (C and D), spatial analogs of some PspGI residues (Ecl18kI residues Cys60 and Arg119) are included for comparison, even though they do not approach the base so closely. In each panel, the structure on the right is 908 horizontally rotated relatively to the one on the left. Only the hydrogen bonds in the planes of the bases are shown. The electron density in (A) and (B) is the 2Fo–Fc ARP/wARP generated map for the preliminary model (contoured at 1 s cutoﬀ). Color coding of DNA and protein molecules is consistent with Figure 2. two cytosines (C-2 and C-1) and their guanine hydrogen bonding partners (G2 and G1) from the minor groove side, and makes direct hydrogen bonds with both guanines via Gln94 (Figure 3). The other PspGI subunit mediates the major groove interactions via the guanidino groups of Arg164 and Arg166, which form two hydrogen bonds each with the outer (G2) and inner (G1) guanines, respec- tively. In addition, Glu165 accepts hydrogen bonds to its terminal carboxylate oxygen atoms from the exocyclic amino groups of the outer (C-2) and inner (C-1) cytosines. If either C-2 or C-1 was N4-methylated, the favorable hydrogen bonding interaction with one of these bases would be replaced by the unfavorable interaction with a non-polar group. As M.PspGI has been classiﬁed as an N4-methyltransferase (2), its protective action against the PspGI restriction endonuclease is accounted for, irrespec- tive of which cytosine is the actual target of methylation. PspGI interactions with the flipped bases Prior modeling studies predicted the interaction of the ﬂipped bases with Phe64 and Arg161 (14). The crystal structure conﬁrms these contacts and identiﬁes additional ones: according to our model, the ﬂipped bases stack against the aromatic rings of Phe64 and the more distal Tyr67 ‘on top’, Gly100 ‘at the bottom’ and the hydropho- bic part of the side chain of Glu60 ‘on the side’ (in the orientation of the left panels of Figure 5A,B). The side walls of the pocket are formed by Ala63 and Ala103, Glu60 and the arginines Arg96 and Arg99, which have their guanidino side chain groups pointing away from the base. In the plane of the bases, the N3 atom of thymine donates a hydrogen bond to the main chain carbonyl oxygen atom of Glu60. The O2 atom of thymine lies within hydrogen bonding distance of the guanidino group of Arg161, but because of poor geometry, it is not clear whether a hydrogen bond is formed. In contrast to the ﬂipped thymine, the ﬂipped adenine interacts with PspGI indirectly through solvent-mediated hydrogen bonds. One solvent molecule is bound to the N7 atom (and possibly the N6 atom) of adenine and also to the carbonyl oxygen atom of Glu60. A second solvent mole- cule is linked to the N1 atom (and possibly the N6 atom) of adenine and also to the guanidino group of Arg99. DISCUSSION A and T flipping by PspGI We have shown here that PspGI ﬂips both bases at the center of its recognition sequence out of the double helix. This makes it the second structure of a restriction enzyme after Ecl18kI to cause this dramatic distortion in DNA. It is thus likely that other restriction endonucleases, which are similar to Ecl18kI and PspGI by primary sequence and cleave related targets analogously, will also be found to ﬂip DNA bases. The DNA distortions caused by PspGI and Ecl18kI share considerable similarity and presumably serve the same purpose—they bring the scissile phosphates closer to a distance that would be found for an endonu- clease with a four base-pair recognition sequence (11). Pocket specificity for pyrimidines PspGI cleaves oligoduplexes with a like-with-like T/T faster than otherwise identical duplexes with a C/C pair (16), suggesting that thymine ﬁts the PspGI pocket better