NKX2.5 HD was overproduced as an MBP-fusion protein. After protease cleavage, the cleaved protein contained an additional Gly residue at the N-terminus, resulting in a molecular weight of 7.6 kDa for the HD. The protein was then subjected to cation-exchange purification, taking advantage of the high pI (calculated pI of 11.4; Gasteiger et al.
); after analysis by SDS–PAGE, it was judged to be over 90% pure (data not shown).
The purified protein was then mixed with double-stranded DNA at a ratio of 1.5 NKX2.5 HD per DNA-binding site in order to ensure full occupation of the DNA-binding sites with NKX2.5 HD protein. The protein–DNA complex was purified by size-exclusion chromatography and the chromatography profile showed that the majority of the protein was present as higher molecular-weight complexes (Fig. 1
a) with an estimated molecular weight of ~30 kDa, corresponding to two HD molecules and one DNA duplex. Uncomplexed protein eluted later as a ~14 kDa protein. This elution time point is earlier than that expected for NKX2.5 HD monomers (7.6 kDa), suggesting the presence of a dimeric form or an elongated shape of the uncomplexed protein. The purity of the protein after the last purification step was estimated to be over 95% based on SDS–PAGE analysis (Fig. 1
Figure 1 Purification of the NKX2.5 HD–ANF −242 DNA complex. (a) Elution profile of the NKX2.5 HD–ANF −242 DNA complex from a Superdex75 column. The estimated molecular weights of a globular protein at time points a and b were ~30 (more ...)
More than ten oligonucleotides of various lengths (8–24) including the ANF −242 motif were used for co-purification and crystallization. Crystals were obtained within 1–3 d, but all showed poor diffraction quality and high mosaicity. To improve the quality of the crystals several additives were added to the reservoir solution, but many reacted with the added reducing agent, thereby hindering the crystallization process. To circumvent this problem, mutant NKX.2.5 HD in which the oxidizable Cys193 was replaced by Ser [NKX2.5 HD (C193S)] was constructed. An NMR experiment showed this protein to have similar binding characteristics as the wild-type NKX2.5 HD (Fodor et al.
). The best-quality crystals were obtained with NKX2.5 HD (C193S) and a 19-mer: a blunt-ended ANF −242 DNA duplex (TGAAGTGGGGGCCTCTTGA/TCAAGAGGCCCCCACTTCA; Fig. 2). The reservoir solution of the optimum crystallization condition for the NKX2.5 HD (C193S)–DNA complex consisted of 50 mM
Tris pH 7.0, 5 mM
, 15% polyethylene glycol monomethyl ether 550. Crystallization drops contained 1 µl sample and 1 µl reservoir solution. The drops were equilibrated against 500 µl reservoir solution using the hanging-drop vapor-diffusion method at room temperature.
A hexagonal crystal of the NKX2.5 HD–ANF −242 complex. The crystal dimensions are 400 × 80 × 80 µm.
The diffraction data for the NKX2.5 HD (C193S)–DNA complex were collected on the APS 22-ID beamline at Argonne National Laboratory from a long rod-shaped crystal that diffracted to 1.7 Å resolution (Fig. 3). The data were initially indexed and scaled in two possible hexagonal space groups: P
22 and P
22. Considering the unit-cell parameters and the molecular weight of the NKX2.5 HD (C193S)–DNA complex, one HD domain and one half of the double-stranded DNA were expected to be present in the asymmetric unit, with a solvent content of 58% (V
= 2.61 Å3
; Matthews, 1968
Diffraction image of a crystal of the NKX2.5 HD–ANF −242 DNA complex obtained using synchrotron radiation at the Advanced Photon Source, Argonne National Laboratory (beamline 22-ID).
After many unsuccessful attempts using various HD structures and search programs including AMoRe
) and MOLREP
(Vagin & Teplyakov, 1997
), the phases for the NKX2.5 HD (C193S)–ANF −242 data were determined by molecular replacement using the MSX-1 HD–DNA complex structure and the program Phaser
(McCoy et al.
). An unambiguous solution was obtained in space group P
= 6.6) but not in P
= 4.6). When the model was positioned in a unit cell, a pair of DNA molecules related by twofold symmetry along the b
axis formed a continuous helix. Subsequent iterative rounds of structural refinement using the rigid-body, simulated-annealing, conjugate-gradient energy-minimization and B
-factor refinement options of CNS
(Brünger et al.
) failed to reduce the crystallographic R
factor below 35% in this space group. The data were then reprocessed in the lower symmetry space group P
and examined for merohedral twinning. The P
6 point group was chosen over the lower symmetry groups P
312 and P
321 because clear systematic absences indicated the presence of a 61
screw axis. Data-processing statistics showed similar R
values for both the P
(6.8%) and P
22 (7.0%) space groups. The detailed data statistics for space group P
are presented in Table 1. Twinning analysis of the data (Yeates, 1997
) in space group P
showed the crystal to be merohedrally twinned with a large twinning fraction (0.47). Subsequent refinement of the NKX2.5 HD (C193S)–DNA structure using the appropriate options in the CNS
program suite (Brünger et al.
) yielded better refinement statistics with R
-factor values (both R
) below 28%. In particular, the maps generated using CNS-TWIN
options in P
showed ordered density for all base pairs. Crystallographic refinement of this structure is in progress.
Crystal data-collection and processing statistics
A high-resolution crystal structure of NKX2.5 HD will provide a detailed picture of NKX2.5–DNA interactions. This will enable the prediction of aberrations in the DNA binding of various mutant NKX2.5 proteins that have been identified in human patients with congenital heart disease.