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					3. Results


3.1 Protein purification and crystallization
    The full length ColE2/Im2 protein complex was very unstable whether in water or
in buffer solutions. It was self-degraded into several small peptide fragments no
matter stored in the room temperature or in the refrigerator. We can obtain tiny
crystals of ColE2/Im2 (Figure 12), but unfortunately these tiny crystals did not
continue to grow to the size big enough for diffraction studies.
    The protein purification elution profiles for nuclease-ColE7/Im7 were shown in
Figure 10 and the purity of the purified protein complex was analyzed by
electrophoretic method in a SDS gel (PhastGel system, Pharmacia, Sweden) (Figure
11). Two clear bands were shown on the gel which were the nuclease-ColE7 and
and Im7, respectively. The purified nuclease-ColE7/Im7 was of high homogeneity,
pure enough for crystallization. The Nuclease-ColE7/Im7 crystals were grown by
vapor diffusion method with sizes between 1.0 x 0.5 x 0.2 mm3 and 1.6 x 0.4 x 0.2
mm3 (Figure 12).


3.2 Structure determination and overall structure of nuclease-ColE7-Im7
    The two complexes of nuclease-ColE7/Im7 with His tag located at different
termini were crystallized in unit cells with similar cell dimensions but different space
groups. The N-terminal His-tagged complex was crystallized in orthorhombic I222,
with the cell constants: a = 62.94 Å, b = 75.25 Å and c = 118.60 Å. The C-terminal
His-tagged complex was crystallized in orthorhombic P21212 with the cell constants: a
= 119.73 Å, b = 62.41 Å and c = 74.14 Å. All types of crystal were soaked in EDTA
buffers to remove divalent metal ions originally associated with the proteins. The
EDTA-treated crystals were then transferred to phosphate buffers containing different
metal ions. Diffraction data of the I222 and P21212 crystals were collected at the
absorption edge of the soaking metal ion using synchrotron radiation or at 1.54 Å
using Cu Kα radiation from a rotating anode (Table 2). Crystals soaked in solutions
containing EDTA, Mn2+, Zn2+or Mg2+ diffracted from 2.6 to 2.0 Å resolution at low
temperature (~100K). The Mg2+-binding site was not found in the Mg2+-soaked
crystals, so the diffraction statistics for Mg2+-soaked crystals were not included in
Table 2.
   The I222 crystals were isomorphous to the previously determined
nuclease-ColE7/Im7, which was therefore used as an initial model for refinement.
The structure of the P21212 crystals was solved by molecular replacement method


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using the I222 structure as the searching model. In the P21212 unit cell, there are two
complex molecules per asymmetric unit (Figure 13), and they are separated by (0.49,
0.52, 0.50) of unit cell dimensions, but not exactly by (0.5, 0.5, 0.5). Therefore the
space group was shifted from the I-center I222 to the primitive P21212, nevertheless,
the protein molecules in the two types of crystals packed in a similar way. The
average rms difference between the two complex molecules in the asymmetric unit of
P21212 cell is only 0.3 Å (for main-chain atoms), therefore, for clarity only the
structure of the first molecule will be used in the following structural comparison.
    The final model of I222 includes one metal ion, one phosphate ion, full length of
Im7 (residues 1-87) and the DNase domain of colicin E7 from residue 447 to 573. The
surface loop region 466 to 473 and the C-terminal residues (574 to 576) were not
observed in the structure due to disorder. The final model of P21212 consists of two
complexes, including two metal ions, two phosphate ions, Im7 (both chain A and
chain B residue: 1-87) and two nuclease-ColE7 (chain A residue: 446-576, chain B
residue: 447-576). The R-factors and the geometric statistics for the final models
were within good ranges as listed in Table 2.


3.3 Comparison between I222 and P21212 structures
    The overall folds of the complex in the two crystal forms are identical (see the
ribbon model in Figure 14). The His-tags were not observed in either I222 or P21212
structures. Im7 contains four α-helices folded in a varied four-helix-bundle structure
nearly identical to the previously determined free-form Im7 (Chak et al. 1996).
Nuclease-ColE7 has a mixed α/β structure with a metal ion bound in a cleft on the
concaved surface. The H-N-H motif (displayed in green in Figure 14) is located at
the C-terminus of nuclease-ColE7 containing two β-strands (βd and βe) and one
α-helix (α5) with a metal ion situated in the center of the motif.
   The P21212 structures in average had lower temperature factors than those of I222
structures. For example, in the Zn-bound P21212 structure the average B-factors
were 30.5 Å2 for nuclease-ColE7 and 23.8 Å2 for Im7, compared to the higher values
of 42.7 Å2 for nuclease-ColE7 and 29.0 Å2 for Im7 in the Zn-bound I222 structure.
In the structures of nuclease-ColE7, the N-terminal end (residues 448 to 460), the
surface loop (residue 486-490) and the long loop between the two β-strands in the
H-N-H motif (residues 446 to 560) had higher B-factors (>40 Å2) in both crystal
forms, indicating that these regions were more flexible (Figure 15). Superimposition
of only the structures of the nuclease-ColE7 in P21212 and I222 gave an average rms
difference of 0.30 Å for the main-chain atoms. However, a larger shift was observed
in the long loop region (residues 550 to 554) within the H-N-H motif with a


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maximum rms difference of 4.7 Å at residue 552. This region corresponded to the
residues with higher temperature factors, therefore, excluding the flexible surface
loops, the two structures are almost identical.


3.4 Metal ions and phosphate binding in H-N-H motif
    The complex crystals were first soaked in EDTA to remove the associated divalent
metal ions. X-ray absorption spectra were recorded for the crystals before and after
EDTA soaking. The absorption at zinc edge was observed before EDTA treatment
and disappeared after ETDA treatment. The previously solved complex structure in
the absence of metal ions was used as the initial model for refinement of the
EDTA-treated crystal (Ko et al. 1999). A Fourier map (Figure 16A) revealed the
absence of a strong peak in the center of H-N-H motif, but instead showed two water
molecules located in the original metal-binding sites. One water molecule was
hydrogen bonded to His545 (ND1). The second water molecule was hydrogen
bonded to the first water molecule and located approximately at the original
metal-binding site. The electron densities of the two histidine residues, His573 and
His544, were ill-defined indicating that the side-chain conformations of the two
residues were not fixed by metal ions. This result demonstrated that the divalent
metal ions initially associated with the complex had been removed by EDTA.
   The EDTA treated crystals were then soaked in phosphate buffers containing Zn2+,
Mn2+ or Mg2+ ions or Sodium Cacodylate buffer containing Ni2+. The diffraction
data for the Mg2+-soaked crystals were collected using the in-house rotating anode
diffraction facility. A magnesium ion was not found in the center of the H-N-H
motif. A Fourier map showing two waters molecules in the center of the H-N-H
motif was obtained, similar to that of the EDTA-treatd crystals (data not shown).
This result indicates that the magnesium ion does not bind in the center of the H-N-H
motif.
    For the transition metal soaked crystals, the X-ray absorption spectra were
recorded first to find out the absorption edges of Zn2+ (1.29004 Å) and Mn2+ (1.89261
Å) ions in crystals. Diffraction data were collected at the absorption edge of the
metal ion correspondingly, to generate the largest anomalous differences between
Friedel’s pairs. Figures 16B and 16C show the Fourier and anomalous difference
maps of Zn2+-bound (in P21212) and Mn2+-bound (in I222) structures at the center of
the H-N-H motif. The electron densities of a metal ion and a phosphate ion were
observed in the Fourier (2Fo-Fc) maps. The locations of metal-binding sites were
further confirmed unambiguously by the anomalous difference maps in which Zn2+
has a strong peak with a peak height of 30 σ and Mn2+ also has a strong peak with a


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peak height of 17 σ. No other metal ion binding sites were identified in the electron
density maps for the Zn2+- and Mn2+-bound crystals. This result demonstrated that
the transition metal ions of Zn2+and Mn2+ bind at the same site in the H-N-H motif,
coordinating to the three histidine residues (His544, His569, and His573) and one
phosphate ion.




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posted:4/2/2013
language:English
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