MATERIALS AND METHODS
Molecular cloning and site-directed mutagenesis. Human Ncb5or
contains 521 amino acid residues that comprise the N-terminal region and
the b5, b5R and CS domains as previously
defined.8 These
segments of the protein contain 50, 113, 111, and 247 residues,
respectively, that also include linker sequences in the middle two
fragments (Figure 1A ). Nomenclature of the constructs used
herein is as follows: N/b5 (Met1 through
Lys137); N/b5-Δ21 (Gly22 through
Lys137); N/b5-Δ34 (Met35 through
Lys137); b5 (Lys51 through
Lys137); and N-term (Met1 through
Leu50). The cDNAs of all Ncb5or constructs (except
N-term) and rice RLF (residues 101-218, XP_015647767) were expressed
with no epitope tag (pET19b or pET22b). The cDNA of N-term was cloned
into the pE-SUMOpro Kan vector (Life Sensors, Malvern, CA), which
contains the 6XHis-SUMO gene immediately in front of the multiple
cloning site previously
described.10Missense point mutants, N/b5-LMAA
(Leu34Ala/Met35Ala) and N/b5-W37A
(Trp37Ala) (Figure 1B ), were generated using
the QuikChange mutagenesis kit (Strategene, La Jolla, CA). All
mutagenesis primers were synthesized by Integrated DNA Technology
(Coralville, IA). All constructs were confirmed by DNA sequencing (ACGT,
Inc., Wheeling, IL).
Protein preparation. Except for N-term, all constructs of human
Ncb5or and rice RLF were expressed in E.coli BL21(DE3) or
BL21(DE3)pLysS or BL21(DE3)pRARE cells and purified as previously
described.8 In
combination with size exclusion chromatography on a Superdex 75 10/300
GL column, ion-exchange chromatography was performed for b5 and N/b5-Δ34
on a Q HP column, N/b5 and variants (N/b5-W37A, Nb5-LMAA, and N/b5-Δ21)
on a SP HP column, and rice RLF on both Q and SP HP columns. Full-length
Ncb5or and its variant Ncb5or-Δ50 were prepared as previously described2 , except that
0.5 mM IPTG was used for induction in TB media at
15oC. All purification steps were performed at
4oC. SDS-PAGE was used to estimate the purity of each
protein used in spectroscopic analyses, kinetics studies and
crystallization screening (all greater than 95%). All purified proteins
were flash frozen in liquid nitrogen and stored as aliquots at
-80oC until use, except for samples prepared for
crystallization screening which were stored at 4oC.
The size of each polypeptide product was confirmed by mass spectrometry.
Expected sizes are: N/b5, N/b5-W37A, N/b5-LMAA, ~15.6
kD; N/b5-Δ21 and RLF, ~ 13.6 kD; N/b5-Δ34, 11.9 kD; and
Ncb5or-b5, 10.2 kD. The ratios of
A413/A280 for purified heme-containing
constructs were as follows: b5 ≥ 4, N/b5 and variants, RLF ≥ 3.6, Ncb5or
~1.0, and Ncb5or-Δ50 ~1.1. The FAD
content of Ncb5or-b5R was determined by A461 and used to
represent enzyme concentrations as previously
described.8 Final
protein yields (mg/L): 8 (b5), 5 (N/b5 and variants), 2 (b5R), ≤1
(Ncb5or, Ncb5or-Δ50, RLF). Solubility in low-salt buffer: N/b5, RLF
> b5 > Ncb5or > Ncb5or-Δ50
> Ncb5or-b5R. Protease inhibitor (Sigma, St. Louis, MO) was
used during protein preparation to prevent proteolytic cleavage at the
b5-CS border of Ncb5or and Ncb5or-Δ50. A SUMOpro Expression System was
used to prepare
N-term.10Briefly, a 6XHis-SUMO-N-term fusion protein was expressed in BL21(DE3)
cells and purified by Ni-NTA affinity resin (Qiagen, Valencia, CA). It
was then cleaved by a 6XHis-SUMO protease11 to release
6XHis-SUMO tag, both of which were removed by nickel resin. The purity
and size of the N-term peptide were confirmed by SDS-PAGE and mass
spectrometry (data not shown).
Spectroscopy. UV/visible spectra were obtained on a Cary 100
Bio spectrophotometer (Varian). The concentrations of oxidized heme in
all constructs were determined with ε413 = 130
mM-1cm-1 as previously
described.2Circular dichroism (CD) analyses were performed on a JASCO-815
spectropolarimeter using protein concentrations of 4-10 µM. Far-UV
(190-250 nm) spectra were recorded using a 1 mm cuvette, whereas a 1 cm
cuvette was used for near-UV (250-350 nm) and visible (350-550 nm) CD
spectra. All CD spectra are reported in units of molar ellipticity
([θ], deg·cm2·dmol-1).
RLF crystallization. A purified RLF construct
(Lys101-Glu218) was concentrated to
44.6 mg/mL in 20 mM Tris pH 8.0 for crystallization screening. All
crystallization experiments were set up using an NT8 drop setting robot
(Formulatrix Inc.) and UVXPO MRC (Molecular Dimensions) sitting drop
vapor diffusion plates at 18oC. 100 nL of protein and
100 nL of crystallization solution were dispensed and equilibrated
against 50 µ L of the latter. Crystals approximately 300-500µ m long that displayed a needle morphology were observed within
one day from the JCSG+ screen (Molecular Dimensions) condition A6 (20%
(w/v) PEG 1000, 100 mM phosphate-citrate pH 4.2, 200 mM lithium
sulfate). A cryoprotectant solution composed of 80% crystallization
solution and 20% (w/v) glycerol was dispensed (2 µ L) onto the
drop, crystals were harvested with a cryoloop immediately and stored in
liquid nitrogen. X-ray diffraction data were collected at the Advanced
Photon Source beamline 17-ID using a Dectris Pilatus 6M pixel array
detector.
Data collection and structure refinement. Intensities were
integrated using
XDS12,
13 via
Autoproc14 and
the Laue class analysis and data scaling were performed with
Aimless15 which
indicated that the crystals belong to the 2/m Laue class with
possible space groups P 2 or P 21. Structure
solution was conducted by molecular replacement with
Phaser16 using
the Ncb5or b5-domain structure (PDB
3LF58 ) as the
search model. The top solution was obtained in the space group P 2
and contained a dimer in the asymmetric unit. Initial refinement with
Refmac17 yieldedR /R free = 42.1%/42.5% and the model was
improved by automated building with
Arp/wARP.18Further refinement and manual model building were conducted with Phenix19,
20 and Coot21 respectively.
TLS parameters 22,
23 were refined in the later stages to
model anisotropic atomic displacement parameters. Disordered side chains
were truncated to the point for which electron density could be
observed. Structure validation was conducted with Molprobity24 and figures
were prepared using the CCP4MG package25 . Polder omit
electron density maps were calculated using
Phenix.26Structure superposition was carried out with
GESAMT.27Crystallographic data are described in Table S1 , and the best
data set was at 1.55 A resolution.