Bacterial Expression and Protein Purification. NsPCS without the 24 N-terminal residues putatively coding for a signal peptide was cloned in pJC40 with a 10His Tag at the
N terminus (20). The recombinant protein was overexpressed in Escherichia coli
BL21 strain, and cells were first incubated at 37°C overnight on 10 ml
of LB medium. The cells were pelleted, washed in sterile water, and
then resuspended into 1 liter of methionine-minus medium from Molecular
Dimensions, supplemented with 40 mg of l-selenomethionine (SeMet), l-lysine, l-threonine, and l-phenylalanine [these three last amino acids create an inhibition of the methionine biosynthesis pathway (21)]. Protein expression was induced at 37°C with 1 mM isopropyl-β-d-thiogalactopyranoside.
After 3 h of growth, cells were pelleted, resuspended in buffer A
[containing 20 mM phosphate (pH 8.0), 500 mM NaCl, 20 mM imidazole, and
1 mM Tris(2-carboxyethyl)phosphine], and disrupted with a French press
at 7 MPa. The resulting soluble fraction was loaded on a nickel-charged
column (HisTrap column, Amersham Pharmacia), and the protein was eluted
by an imidazole step gradient (150 mM wash and 500 mM elution). Because
NsPCS precipitated in buffer A after 2 h at room temperature or during
the following concentration steps, the protein was then exchanged into
buffer B [50 mM Mes (pH 5.5), 50 mM NaCl, and 1 mM
Tris(2-carboxyethyl)phosphine] and concentrated up to 10 mg·ml–1 before crystallization trials. The yield of SeMet NsPCS was typically of 5 mg per liter of culture. The incorporation of
SeMet was confirmed by MALDI mass spectrometry.
All of the crystallization experiments were carried out at 293 K by
using the hanging drop vapor diffusion technique. Optimal
crystallization conditions were obtained by a microseeding technique.
The best crystals of the native protein were obtained by mixing 2 μl of
SeMet-labeled NsPCS with 2 μl of a reservoir solution (1 ml) containing
100 mM Mes (pH 5.0), 15–20% (wt/vol) PEG 4K, and 100–400 mM NaCl. Thin
plates (approximate dimensions: 10 × 100 × 100 μm3) grew in ≈2 days and belong to space group P21 with unit-cell parameters as follows: a = 47.86 Å, b = 62.47 Å, c = 76.55 Å, and β = 101.38° (see Table 1, which is published as supporting information
on the PNAS web site). Crystals of the acyl–enzyme were obtained at
acidic pH by mixing 2 μl of SeMet-labeled NsPCS with 0.2 μl of a 200 mM
GSH solution and 2 μl of a reservoir solution containing 100 mM citric
acid (pH 2.6–3.4), 100 mM NaCl, and 10–20% (wt/vol) PEG 4K. Although
crystals have the same shape as the native crystals, they grow in ≈2
weeks and correspond to a different crystal form (Table 1).
Data Collection and Phasing.
Native and acyl–enzyme crystals were soaked 1 min in mother liquor
containing 30% glycerol and 50% ethylene glycol, respectively, and then
directly flash-frozen in liquid nitrogen before data collection.
Diffraction data on the native crystal were collected at three
wavelengths at beamline BM30 (European Synchrotron Radiation Facility,
Grenoble, France) by using a 180-mm MAR CCD detector, and diffraction
data on the acyl–enzyme crystal were collected at beamline ID29 on an
ADSC Q210 2D detector. Data sets were processed with mosflm (22) and scaled by using scala from the ccp4 suite (23, 24).
Structure Determination and Refinement. Ten selenium positions were found from the MAD data by the program solve (25).
From these positions the NCS operators for a dimer could be derived,
and, subsequently, 70% of the molecule was automatically built by the
program. Several rounds of iterative model building and refinement were
performed by using the programs turbo (26) and refmac (27).
The final native model contains two molecules (consisting of amino
acids 29–239), 259 solvent molecules, and two ions assigned as calcium.
Electron density is missing for the His-tag and the four N-terminal
residues as well as the three C-terminal residues.
The acyl–enzyme structure was solved by molecular replacement by using the program molrep (28) with the native dimeric structure as a search model. After several cycles of model building and refinement with refmac,
the final model displays the same disordered regions as the native
model, with additional disorder for residues 87–94, 106–108 in molecule
A, and 29–31 and 88–91 in molecule B. Additionally, the main chain and
side chain of Arg-180 (a Ramachandran outlier in the native structure)
display a large disorder in both molecules and have been modeled as a
glycine. The final model contains two molecules, 391 solvent molecules,
and four ions (assigned as two calcium and two chlorides). For both
structures, all of the residues fall in the favored region of the
Ramachandran plot except for the above-mentioned Arg-180. procheck (29)
was used to assign secondary structure and revealed that structural
parameters were consistently better than or within the average for
structures at comparable resolution. Graphics presented in this article
were generated by using the program pymol (www.pymol.org).