Initial attempts to purify recombinant full-length ZEBRA indicated that the protein was readily degraded by endogenous E. coli proteases. Limited proteolysis with trypsin yielded a partially stable 8 kDa fragment, which was determined by N-terminal sequencing and mass spectrometry to correspond to the C-terminal 71 residues of ZEBRA (data not shown). Accordingly, we made a construct spanning this region, ZEBRA175–245, which includes the bZIP domain. This fragment was expressed in E. coli BL21(DE3) cells, yielding ~0.5 mg of highly purified protein per litre of bacterial culture (Fig. 1, lane 2). The fragment elutes from a Superdex-75 HR10/30 gel-filtration column as a single peak corresponding to an apparent molecular weight of 15 kDa (Fig. 2
a) or approximately twice the calculated molecular weight of monomeric ZEBRA175–245 (8380 Da). This indicates that ZEBRA175–245 is homodimeric in solution, consistent with the presence of the bZIP domain. The existence of a homodimer was confirmed by a cross-linking study using ethylene glycol bis(succinimidyl succinate) (EGS; Fig. 2
b). Purified ZEBRA175–245 has a tendency to aggregate in solution and we were unable to concentrate it to concentrations higher than ~3 mg ml−1. Attempts to crystallize ZEBRA175–245 in complex with oligonucleotide duplexes of various lengths resulted in several crystal forms (e.g. Fig. 3
a); however, none of these were suitable for structure determination.
Purification of ZEBRA constructs. 12% SDS–PAGE showing molecular-weight markers (kDa) in lane 1, ZEBRA175–245 in lane 2 and ZEBRA175–236/mut in lane 3. Approximately 5 µg of protein are loaded in lanes 2 and 3.
Figure 2 Evidence for dimer formation. (a) Analytical gel-filtration profile of ZEBRA175–245 on a Superdex-75 HR10/30 column. Elution volumes of molecular-weight standards are indicated. (b) Cross-linking study with EGS. ZEBRA175–245 (2.5 µg) (more ...)
Figure 3 Crystals of ZEBRA C-terminal constructs in complex with DNA. (a) ZEBRA175–245 (b) ZEBRA175–236/mut. In both cases cocrystallization was with the same 19-mer DNA duplex (see §2). The scale bar corresponds to 0.2 mm.
We therefore began crystallization trials with a modified construct, ZEBRA175–236/mut
, obtained by deleting nine C-terminal residues (VLHEDLLNF) and introducing two point mutations, S186A and C189S. The deletion eliminates five hydrophobic residues and considerably increases protein solubility, allowing us to concentrate the purified protein to >10 mg ml−1
. The deletion does not appreciably alter the DNA-binding affinity, as measured in an electrophoretic mobility shift assay (not shown). The C189S mutation stabilizes the protein against oxidation and corresponds to the mutation which yielded improved crystals of Fos-Jun-DNA (Glover & Harrison, 1995
), while the S186A mutation renders the ZEBRA sequence more similar to that of Fos and Jun. The typical degree of chemical purity obtained is illustrated in Fig. 1 (lane 3). The homogeneity of the protein and of protein–DNA complexes was confirmed by mass spectrometry (data not shown). Crystallization trials with ZEBRA175–236/mut
and DNA duplexes of various lengths yielded several promising crystal forms. One crystal form, obtained in complex with a 19-mer DNA duplex and characterized by a needle-like morphology (Fig. 3
), reproducibly diffracted synchrotron radiation to better than 3 Å resolution (Fig. 4).
Figure 4 Diffraction pattern of a ZEBRA175–236/mut–DNA crystal. The crystal was exposed at 100 K after soaking in 25% PEG 400 for cryoprotection. The rotation angle used was 1°, the crystal-to-detector distance was 150 mm (more ...)
Using the microfocus beamline ID13 at the ESRF, a complete 2.5 Å data set could be collected from a single frozen crystal by successively exposing different crystal volumes to X-radiation. Data processing was carried out in space group C
2 using XDS
). Data-collection statistics are summarized in Table 1. Packing-parameter calculations suggest that the asymmetric unit contains one ZEBRA175–236/mut
homodimer bound to one DNA duplex. This corresponds to a Matthews coefficient (Matthews, 1968
) of 2.3 Å3
, with a solvent content of 45%. Self-rotation functions show the consistent presence of a strong peak (>50% of the origin peak) at polar angles ϕ = 142, ψ = 90° for all values of the polar angle κ between 0 and 180°, corresponding to an axis of symmetry parallel to the ac
plane (Fig. 5
). A scan along the κ angle displays a local maximum at κ = 35° (Fig. 5
). This is close to the mean helical twist of 36° between successive base pairs for B-DNA, suggesting that the DNA axis may be oriented in this direction.
Data-collection and processing statistics
Figure 5 Self-rotation function calculated using GLRF (Tong & Rossmann, 1997 ) with an integration radius of 15 Å and data in the resolution range 30–2.5 Å. (a) The κ = 36° section. Contour (more ...)