6110 Nucleic Acids Research, 2008, Vol. 36, No. 19 Figure 1. PspGI target sequence. (A) Oligonucleotide duplex used for co-crystallization with the D138A variant of PspGI. The recognition sequence is marked by the yellow box, and the symmetry breaking base pair is highlighted in orange. (B) Comparison of the hydrogen bonding requirements of a T/A (upper left) and A/T pair (lower left) with those of a G/C (upper right) and C/G pair (lower right). guanine or cytosine. Direct biochemical evidence for ade- nine or thymine ﬂipping by PspGI/EcoRII is diﬃcult to provide, but ﬂuorescence experiments with 2-aminopurine (2AP) demonstrate that this adenine analog (which shares some hydrogen bonding requirements with guanine) can be ﬂipped by PspGI/EcoRII (12,13). Evidence for ﬂipping of a natural base in the CCNGG context by PspGI has been obtained for cytosine: this base in the central position of the CCNGG sequence was found to deaminate faster in the presence of the enzyme (14). Moreover, the accessibil- ity of this base to chloroacetaldehyde modiﬁcation in the PspGI-DNA complex provides additional evidence that cytosine can be unstacked by PspGI (15). At present, it is still unclear how PspGI and EcoRII distinguish between W and S at the center of their recognition sequence, and whether the lack of activity against CCSGG containing DNA goes hand-in-hand with a lack of ﬂipping of at least one base of the central G/C pair. Here, we report the crystal structure of PspGI in complex with target DNA (with the CCWGG sequence). The structure pro- vides a crystallographic demonstration that PspGI ﬂips both adenine and thymine in its target sequence. In com- bination with biochemical results (16), it also sheds light on the speciﬁcity of the enzyme for the nucleotides at the center of its recognition sequence. MATERIALS AND METHODS PspGI purification PspGI-D138A was puriﬁed from ER2744 (fhuA2 glnV44 e14 rfbD1 relA1 spoT1 endA1 thi-1 (mcrC-mrr) 114::IS10 lacZ::T7gene1) cells containing pET21a-PspGI- D138A. Cultures were allowed to grow to an OD600 of 0.4 then induced with 0.3 mM isopropyl b-D-1-thiogalacto- pyranoside (IPTG) for 12 h at 168C after which they were spun down and frozen at 708C. The cell pellets were thawed on ice and re-suspended in lysis buﬀer (20 mM Tris pH 8.5, 50 mM NaCl) containing protease inhibitors (Complete EDTA-free Protease Inhibitors, Roche Diagnostics GmbH, Mannheim, Germany). Cells were broken by sonication followed by centrifugation at 20 000g for 30 min to remove cell debris. The supernatant was heated to 708C for 30 min followed by centrifugation at 20 000g for 30 min to remove precipitated proteins. The supernatant was loaded onto a 14 ml P11 column (Whatman, Maidstone, UK) and washed with 3 column volumes of wash buﬀer (20 mM Tris pH 8.5, 50 mM NaCl). PspGI-D138A was eluted from the column with elution buﬀer (20 mM Tris pH 8.5, 300 mM NaCl) and spin concentrated using Millipore 10 000 MWCO spin col- umns (Billerica, MA, USA). The concentrated protein was dialyzed into dilution buﬀer A (10 mM Tris pH 7.9, 50 mM KCl, 50% glycerol) and stored at 208C. Protein concen- trations were determined by a modiﬁed Bradford assay (Bio-Rad, Hercules, CA, USA) and purity was judged by SDS–PAGE. Puriﬁcation of PspGI-D138A labeled with seleno- methionine followed a similar strategy except that expres- sion was in B834 (F – ompT hsdS gal dcm met DE3). Cultures were grown in 500 ml M9 minimal media supple- mented with 5% LB and 50 mg/ml carbenicillin at 378C until an OD600 of 0.7 was reached. Cells were spun down at 1000g and re-suspended in M9 minimal media. The cells were then grown for 2 h at 378C in M9 minimal media with 50 mg/ml carbenicillin to use up any remaining L-methionine. After 2 h, L-selenomethionine (Acros Organics, Geel, Belgium) was added to 50 mg/ml and IPTG was added to 0.3 mM and PspGI-D138A was expressed for 10 h. Puriﬁcation followed the same strategy as for unlabeled protein except that 1, 4-dithiothreitol (DTT) was added to all buﬀers to 1 mM. Quantiﬁcation and purity estimation were the same as above and the MALDI-TOF spectrum was consistent with incorporation of selenomethionine. PspGI crystallization HPLC grade oligodeoxynucleotides 50-CATCCAGG TAC-30 (oligo 1) and 50-GGTACCTGGAT-30 (oligo 2) obtained from Metabion (Martinsried, Germany) were dissolved in 10 mM Tris pH 8.0, mixed in 1:1 molar ratio, heated to 958C and cooled slowly overnight to 48C. The (inactive) D138A mutant of the PspGI protein and its selenomethionine variant were concentrated in 10 mM Tris pH 7.5 and 50 mM KCl to 14.4 mg/ml and 16.4 mg/ml, respectively. Annealed oligoduplex and concentrated protein were mixed in stoichiometric ratio (one oligoduplex per PspGI dimer), supplemented with MnCl2 (ﬁnal concentration of about 6 mM) and incubated at room temperature for 1 h prior to crystallization. Crystals were grown by the vapor diﬀusion technique. Sitting drops were set up automatically using a Lissy robot (Zinsser Analytic, Frankfurt, Germany) for pipet- ting reservoir buﬀers and a MicroSys 4000 robot (Genomic Solutions, Ann Arbor, Michigan, USA) for pipetting crystallization drops (at the 200 + 200 nl scale). The preliminary crystallization condition [20% MPD (2-methyl-2,4-pentandiol), 0.1 M citric acid, ﬁnal pH 4.0] was scaled up to the 2 ml (protein) + 2 ml (buﬀer) and optimized by the addition of 0.4 ml of 0.1 M cobalt (II) chloride. Small crystals appeared overnight. Crystal growth lasted for about a week and then reached