P.-C. Guo et al. / Biochimica et Biophysica Acta 1844 (2014) 1486–1492
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Table 1
2. Materials and methods
Data-collection statistics for the crystal of yeast Gre2 and the Gre2–NADPH complex.
2.1. Overexpression and purification of Gre2 and mutants
Gre2
Gre2–NADPH complex
Data collection
Space group
Unit cell (Å, °)
90, 90, 90
The coding sequence of GRE2/YOL151W was amplified by PCR, using
S. cerevisiae S288c genomic DNA as the template, and cloned into a
pET28a-derived vector. This construct adds a hexahistidine-tag to the
N-terminus of the recombinant protein, which was over-expressed in
an E. coli BL21 (DE3) (Novagen, Madison, WI) strain using a 2 × YT
culture medium. The cells were induced with 0.2 mM isopropyl-β-D-
thiogalactoside (IPTG) at 16 °C for 20 h when OD600nm reached 0.6.
Cells were harvested by centrifugation at 8000 g for 10 min and resus-
pended in lysis buffer (20 mM Tris–HCl, pH 7.0, and 200 mM NaCl).
After 5 min of sonication and centrifugation at 12,000 g for 25 min,
the supernatant containing the soluble target protein was collected
and loaded to a Ni-NTA column (GE Healthcare) equilibrated with bind-
ing buffer (20 mM Tris–HCl, pH 7.0, and 200 mM NaCl). The target
protein was eluted with 250 mM imidazole buffer and further loaded
onto a Superdex 75 column (GE Healthcare) equilibrated with 20 mM
Tris–HCl, pH 7.0, and 50 mM NaCl. Fractions containing the target pro-
tein were pooled and concentrated to 20 mg/mL by ultrafiltration
(Millipore, 10 kDa cut-off). The purity of the protein was estimated on
SDS-PAGE and the protein sample was stored at −80 °C. The mutant
proteins were expressed, purified, and stored in the same manner as
the wild-type protein.
P212121
P212121
48.72, 97.85, 139.00
90.31, 92.89, 201.09
90, 90, 90
Molecules per asymmetric unit
Resolution range (Å)a
Unique reflections
Completeness (%)
bI/σ(I)N
2
4
50.00–2.00 (2.03–2.00) 50.00–2.40 (2.44–2.40)
45,207 (22,08)
99.2 (97.1)
18.07 (6.32)
7.9 (20.7)
67,436 (3,332)
99.3 (100.0)
15.93 (3.52)
8.9 (46.3)
Rmergeb (%)
Average redundancy
4.5 (4.0)
5.6 (5.7)
Structure refinement
Resolution range (Å)
(2.05–2.00)
50.00–2.00
50.00–2.40
(2.46–2.40)
R-factorc/R-freed (%)
Number of protein atoms
Number of water atoms
RMSDe bond lengths (Å)
RMSD bond angles (°)
Mean B factors (Å2)
22.4/26.6
5380
346
24.2/28.8
10194
128
0.008
1.044
56.9
0.007
1.105
50.9
Ramachandran plotf
Most favored (%)
Additional allowed (%)
PDB ID
98.4
1.2
98.3
1.3
a
2.2. Crystallization, data collection, structure solution, and refinement
Values in parentheses are for the highest resolution shell.
Rmerge = ∑hkl∑i|Ii(hkl) − bI(hkl)N| / ∑hkl∑i|Ii(hkl)|, where Ii(hkl) is the intensi-
b
ty of an observation and bI(hkl)N is the mean value for its unique reflection; summations
are over all reflections.
Crystals of Gre2 were obtained at 289 K using the hanging drop vapor-
diffusion techniques, with the initial condition of mixing 1 μL of the
20 mg/mL protein sample with an equal volume of mother liquor (25%
polyethylene glycol 2000 MME, 0.2 M (NH4)2SO4, and 0.1 M sodium ace-
tate, pH 4.6). The crystals grew to approximately 0.1 × 0.2 × 0.4 mm in
about 3 days. Crystals of the Gre2–NADPH complex were obtained by
co-crystallizing Gre2 with 5 mM NADPH in the same mother liquor. The
crystals were transferred to a cryoprotectant [reservoir solution supple-
mented with 25% (v/v) glycerol] and flash-cooled at 100 K in liquid nitro-
gen. Both data were collected at a radiation wavelength of 0.9795 Å at the
Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied
Physics, Chinese Academy of Sciences, using a beamline BL17U at 100 K
with a Q315r CCD (Marresearch). Data processing and scaling were per-
formed using the HKL2000 package [19]. Both crystal structures were de-
termined by the molecular replacement method with MOLREP [20] using
the coordinates of Sporobolomyces salmonicolor aldehyde reductase (PDB
ID: 1Y1P, sequence identity is 30%) as the search model. Refinement was
carried out using REFMAC [21] and Coot [22]. The overall assessment of
model quality was performed using MolProbity [23]. The crystallographic
parameters of the structures are listed in Table 1. All structural figures
were prepared with PyMOL [24].
c
R-factor = ∑h||Fo(h)| − |Fc(h)|| / ∑h|Fo(h)|, where Fo and Fc are the observed and
calculated structure-factor amplitudes, respectively.
d
R-free was calculated with 5% of the data excluded from the refinement.
Root-mean square-deviation from ideal values.
Categories as defined by MolProbity.
e
f
100 mM phosphate buffer, pH 7.5, 0.2 mM NADPH, purified protein
and substrates at various concentrations. Isovaleraldehyde was
dissolved in absolute alcohol (final concentration of alcohol in assay
was 1%). The reactions were triggered by adding the purified protein
solution, and the decrease in absorbance at 340 nm (εNADPH = 6220
M−1 cm−1) was monitored with
a DU800 spectrophotometer
(Beckman Coulter) equipped with a cuvette holder fixed at 30 °C. The
decrease of absorbance resulting from the non-enzymatic reduction of
substrates upon addition of NADPH was set as background control for
each assay.
3. Results and discussion
3.1. Overall structure
2.3. Computational docking
An asymmetric unit of Gre2 contains two subunits, with an overall
root mean square deviation (RMSD) of 0.26 Å over 316 Cα atoms
(Fig. S1A). The two subunits have a buried interface of 1050 Å2 that
might be due to the crystal packing, because the result of PISA [27] eval-
uation and the analysis of gel filtration chromatography demonstrated
that Gre2 was monomeric (Fig. S1B). A Gre2 molecule can be divided
into two distinct domains: an N-terminal cofactor-binding domain
and a C-terminal substrate-binding domain (Fig. 1A). The cofactor-
binding domain adopts a Rossmann-fold motif [28], with a parallel
seven-stranded-sheet sandwiched by a total of six-helices (α1–5, and
The three dimensional structure of isovaleraldehyde was trans-
formed with the program phniex.elbow [25] from the simplified molec-
ular input line entry specification (SMILES) level representations CC(C)
CC_O. Docking runs were performed using the program HADDOCK
[26], based on our Gre2–NADPH structure. Up to 15 initial clusters
were generated, for each of which multiple conformations were scored.
The best scoring pose was a rigid-body minimized and scored for
electrostatic and van der Waals interactions.
α7). Notably, the dinucleotide-binding motif (G7A8N9G10F11I12A13)
2.4. Enzymatic activity assay
locates at the region between β1 and α1. The substrate-binding domain
has four α-helices (α6 and α8–10) and one twisted β-sheet, formed by
β1′, β2′, and β3′, located so as to cover the bound NADPH.
The kinetic parameters of native Gre2 and its mutants were mea-
sured as previously described [17] with minor changes. All assays
were performed at 30 °C in a standard assay mixture containing