Protein Expression and Crystallography
DIM-5 protein was expressed and purified as described (Zhang et al., 2002
). For cocrystallization, an H3 peptide (residues 1–15) was added at a final concentration of 2 mM to purified DIM-5 protein (12 mg/ml in 20 mM glycine [pH 9.8], 150 mM NaCl, 5 mM DTT, 5% glycerol, and 600 µM AdoHcy). Crystals were obtained using the hanging drop method at 16°C, with mother liquor containing 0.1 M Tris (pH 8.4–8.6), 20%–25% polyethylene glycol 2000 monomethyl ether, 0.2 M trimethylamine, and 5 mM DTT.
X-ray data from a single frozen crystal were collected on an ADSC Q315 CCD detector at beamline X25 at the National Synchrotron Light Source, Brookhaven National Laboratory. The exposure time for a 1° rotation was 120 s at 1.0Å wavelength with 400 mm detector-to-sample distance. Data acquisition and processing for a total of 135° rotation used the HKL2000 software package (Otwinowski and Minor, 1997
). Crystallographic data statistics are shown in . Data from 10.0–4.0 Å were used in the structure solution by molecular replacement. All data to 2.6 Å were used for refinement.
Summary of X-Ray Diffraction Data
The coordinates of substrate-free DIM-5 (PDB 1ML9) were used to search the molecular replacement solution using the program AMoRe (Navaza, 2001
). With reference to the search model, two solutions were found: the orientation of the DIM-5 molecule in space group P21
corresponds to Eulerian rotations of (103.07°, 80.86°, 0.15°) and (95.48°, 45.64°, 109.30°), with translations along a, b, and c axes of (0.384, 0.0547, 0.0630) and (0.0966, 0.6179, 0.1581) in fractional coordinates, respectively. The solutions, with the correlation coefficient of 0.488 and R factor of 0.44, indicated each asymmetric unit contains two complexes. The contact between the two DIM-5 molecules is mediated through N-terminal residues 30–45.
The resulting model, optimized by rigid-body refinement of X-PLOR (Brünger, 1992
), provided an initial phase that was further improved by an overall anisotropic B factor optimization (B11 = 20.7, B22 = 17.5, and B33 = 19.9) and a bulk solvent correction (X-PLOR), resulting in a R factor of 0.33 and R free of 0.37. The difference Fourier maps (2Fobs
), phased from the protein model, were then calculated at 3.0, 2.8, and 2.6 Å , respectively, and inspected using the graphic program O (Jones and Kjeldgard, 1997
). Electron densities, without 2-fold noncrys-tallographic symmetry averaging, were clearly visible in both molecules corresponding to the AdoHcy, the zinc coordinated by post-SET Cys residues, and the structured portion of the H3 peptide. These segments were positioned manually to fit the electron density. One cycle (100 steps) of least-squares positional refinement using meaX- PLOR gave an R factor of 0.26 and R free of 0.33. Several cycles of least-squares refinement of positional and individual B factors, followed by manual model building using O, were carried out. The noncrystallographic symmetry restraints were imposed on the two complexes during the refinement (with NCS weight of 300). A series of simulated annealing omit maps were used to guide the manual model fitting.
At this stage of refinement, we realized that the disordered ends of the peptide, residues 13–15 or 1–6, colocalize to the general area of the beginning or the end of disordered protein residues 286–304, respectively (). Discontinued densities do exist, but we were not able to unambiguously distinguish between the peptide and protein densities. The assignment of solvent molecules to these densities would reduce the difference between values of R factor and R free; but we took a conservative approach without including such solvent molecules in the final model (with R factor of 0.22 and R free of 0.32).
Besides residues 286–304 between the SET and post-SET regions, two other segments of DIM-5 were not modeled in the final structure: the N-terminal residues 17–25 and residues 90–96 of the pre-SET domain. In addition, a few stretches of residues (52–61, 85–89, and 97–98 of pre-SET, and 190–202 and 212–224 of SET) are flexible, as indicated by disordered side chains and relatively higher crystallographic thermal factors of >75 Å2 (2–3 times higher than the rest of the protein). As a result, many side chains of residues within or near the flexible stretches were modeled only as alanine (pre-SET residues: K53, N54, Q60, V64, S70, E72, E73, and D83; SET residues: E181, S185, E186, E194, S195, T196, R199, R200, D215, S216, L217, L221, and E227). The flexible stretches are clustered together in the folded structure: for example, the loop after strand β3 (K53, N54) is next to strand β9 (E181) and helix αF (S185 and E186); two 310 helices, αA (Q60) and αI (L221), are packed together.
As noted, the pre-SET domain contains nine invariant cysteine residues that are grouped into two segments of five and four cysteines separated by a disordered region (residues 90–96). We noticed that the first Cys segment (residues 50–99) is more mobile (with an average thermal value of 70 Å2
) than the second segment (residues 100–150) with an average thermal value of 40 Å2
. This observation suggests an intriguing possibility that the zinc can be transferred from pre-SET triangular cluster to the post-SET domain, analogous to methallothiomeins containing two metal clusters (Jacob et al., 1998
). The dynamic nature of the pre-SET domain is confirmed by a second data set, from a different crystal, collected at beamline 17-ID of the Advanced Photon Source, Argonne National Laboratory. This time we refined the structure using tighter restraints on NCS (weight = 700, instead of 300 used in the previous refinement). The tighter NCS restraints resulted in a smaller difference between R factor (0.26) and R free (0.32) (again, no water molecules were included) at resolution range of 10–2.8 Å (28,713 reflections). However, the resulting structure is nearly identical to the previous one, particularly in the local regions around the active site.