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PROTEINS: Structure, Function, and Genetics 28:285–288 (1997)
Expression, Crystallization and Preliminary X-Ray
Diffraction Study of FtsY, the Docking Protein
of the Signal Recognition Particle of E. coli
Guillermo Montoya,1 Cecilia Svensson,1 Joen Luirink,2 and Irmgard Sinning1*
1European Molecular Biology Laboratory, Structural Biology Programme, Heidelberg, Germany
2Department of Microbiology, Institute of Molecular Biological Sciences, Biocentrum Amsterdam, DeBoelelaan 1087,
Amsterdam, The Netherlands
ABSTRACT
FtsY is the docking protein or
SRa homologue in E. coli. It is involved in
targeting secretory proteins to the cytoplasmic membrane by interacting with the signal
recognition particle, controlled by guanosine
58-triphosphate. Two different constructs have
been used in crystallization studies: the fulllength protein and a truncated fragment with
a his-tag at the C terminus. Only the second
construct resulted in crystals suitable for x-ray
diffraction. The crystals belong to the monoclinic space group P21 with cell dimensions a 5
32.20 Å, b 5 79.57 Å, c 5 59.21 Å, and b 5 94.45,
and contain one molecule per asymmetric unit.
At cryogenic temperatures the crystals diffract to
a resolution limit of 2.5 Å by using a rotating
anode, and beyond 1.8 Å by using synchrotron
radiation. Proteins 28:285–288, 1997.
r 1997 Wiley-Liss, Inc.
Key words: SRP; SRP receptor; GTPase; expression; crystallization; x-ray diffraction
INTRODUCTION
Targeting and translocation of proteins into and
across membranes in eukaryotes and prokaryotes, is
mediated by hydrophobic N-terminal signal sequences. This suggests similarities in their basic
mechanisms of targeting and translocation.1 In mammalian cells, targeting of most proteins to the endoplasmic reticulum is mediated by the SRP, which
consists of a 7S RNA and six polypeptides of 9, 14, 19,
54, 68, and 72 kDa.2 The SRP binds via its 54-kDa
subunit (SRP54) to the signal sequence of the nascent polypeptide chain as soon as it becomes available at the ribosome, thereby arresting elongation.3
The complex formed by the ribosome, the nascent
polypeptide chain, and the SRP is targeted to the
endoplasmic reticulum by interacting with a docking
protein complex (or SRP receptor) formed by two
proteins. Both the SRa and the SRb have GTPase
activity.4,5 The SRP and the receptor are released
after GTP hydrolysis, and the remaining ribosomer 1997 WILEY-LISS, INC.
nascent chain complex interacts with a complex of
membrane proteins, which catalyzes membrane insertion and translocation of the nascent chain.
In E. coli, several factors of the general secretory
pathway have been identified using genetic and
biochemical approaches.6 Molecular chaperones, such
as SecB, function to maintain certain precursor
proteins in a translocation competent conformation
in the cytosol and to target them to the cytoplasmic
membrane where a complex composed of SecA/Y/E/G
and SecD/F assists in membrane insertion and translocation. The role of the SRP in E. coli is poorly
understood. It is much simpler than the eukaryotic
SRP consisting of only one protein—p48, Ffh—and a
4.5 S RNA.7,8 SRP may support cotranslational translocation in a separate secrectory pathway or could
form part of the above-mentioned general secretory
pathway. The first hypothesis is supported by identification of E. coli FtsY as the homologue of SRa. FtsY
displays significant sequence similarity to SRa9,10
and was shown to be essential for efficient secretion
of a subset of preproteins.11 Furthermore, the GTPdependent interaction of Ffh and FtsY was demonstrated,12 and it was shown that Ffh and FtsY act as
regulatory proteins for one another.13 Earlier, a role
in cell division was proposed based on the location of
ftsY in an operon together with ftsE and ftsX, in
which mutations have been identified that affect cell
division.14 FtsY consists of 497 amino acids (54 kDa)
and behaves as a monomer in solution—although it
displays an unusual electrophoretic behavior. FtsY
consists of three domains: an acidic N-terminal
domain, an N domain (named in analogy to the N
domain in Ffh and SRP54) and a C-terminal G
domain, which contains the consensus elements for
GTP binding. Together with SRP54, Ffh, the SRa
Abbreviations: SRP, signal recognition particle; Ffh, fifty-four
homologe; DTT, dithiothreitol; IPTG, isopropyl-thio-bd-galactopyranoside; GDP, guanosine diphosphate; GMP-PNP, guanylylimidodiphosphate; MAD, multiple wavelength anomalous
diffraction; Se-Met, selenomethionine; GTP, guanosine triphosphate
*Correspondence to: Irmgard Sinning, EMBL, Meyerhofstr.
1, D-69117. Heidelberg, Gemany.
Received 20 September 1996; Accepted 11 November 1996
286
G. MONTOYA ET AL.
and SRb proteins, FtsY forms a distinct family of
GTPases.15 So far, no high-resolution structural information is available for any member of the SRP
family of GTPases. In this report we describe the
expression and crystallization of the full-length protein and of the NG domain of FtsY.
MATERIALS AND METHODS
Expression and Purification
Expression and purification of recombinant FtsY
have been performed in principle as described earlier.11 Some modifications to improve the expression
and purification of the protein have been included,
for example, the application of a molecular sieve
column after the ionic exchange step. Besides the
full-length protein, the NG fragment of FtsY containing the residues 197 to 497 and 6 histidine residues
at the C terminus was expressed in E. coli, strain
BL21(DE3)pLys S (Novagen), by using the pET9
plasmid. Cells were transformed by electroporation,
and colonies were selected by double resistance
against kanamycin (30 µg/ml) and chloramphenicol
(34 µg/ml). Cells were grown in LB medium supplemented with 0.4% glucose plus kanamycin and chloramphenicol. Cells were induced with 0.4 mM IPTG
at OD600 5 0.3. After 2 hours the cells were collected
by centrifugation and stored at 280°C until use. The
cells were disrupted by two passes through a french
press in 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10
mM MgCl2. Unlysed cells were removed by centrifugation and 1 mM GDP was added to the supernatant.
The sample was loaded on a nickel column (Chelating Sepharose Fast Flow, Pharmacia) equilibrated
with 50 mM Tris-HCl pH 7.4, 300 mM NaCl, and 10
mM MgCl2. The column was first washed with a
gradient from 0 to 100 mM imidazol and then a
gradient from 100 to 800 mM imidazol was applied.
The protein eluted as a single peak around 300 mM
imidazol. The sample was concentrated to 8 mg/ml,
and then subjected to dialysis against 50 mM TrisHCl pH 8.0, 200 mM (NH4)2SO4, 0.2 mM ZnCl2, 5mM
MgCl2, 5 mM DTT for 1 hour. GDP was exchanged by
GMP-PNP following the procedure described by John
et al.16 The GMP-PNP exchanged sample was then
subjected to gel filtration chromatography using a
Superose 12 column (Pharmacia) with a mobile
phase of 10 mM Tris-HCl pH 7.4, 100 mM NaCl, and
10 mM MgCl2. The protein eluted as a single peak
followed by the excess of GMP-PNP. The nucleotide
content of the protein before and after exchange was
monitored by HPLC analysis.16
Seleno-methionine (Se-Met) containing NG fragment was produced using the E. coli methionine
auxotroph strain B834 (DE3). Cells were transformed by electroporation, and colonies were selected by using kanamycin. The procedure was similar to the selection procedure described above. A
preculture was grown overnight at 37°C in minimal
medium plus methionine (40 mg/ml) and kanamycin.
The preculture was inoculated in 1l minimal medium plus 40 mg/ml methionine and kanamycin and
incubated at 37°C until it reached 0.8 OD at 600 nm.
The cells were collected by centrifugation, and the
pellet was dissolved in 1 l minimal medium plus
kanamycin but without methionine. The culture was
incubated overnight at 30°C. In the morning Se-Met
was added (40 mg/ml), and the culture was incubated for 1 hour at 37°C and then induced with 0.4
mM IPTG. After 4 hours, the cells were collected and
stored at 280°C until use. The purification and
crystallization procedure for the Se-Met-containing
protein was the same used for the native protein.
The purity of the full-length protein, the NG domain,
and the Se-Met-NG domain were checked by SDSPAGE and mass spectrometry, and was higher than
98%.
Crystallization
The full-length protein and the NG fragment of
FtsY were concentrated to 5 and 10 mg/ml, and
crystallization experiments were performed by using
the hanging drop vapor diffusion method at 20 and
4°C using Linbro 24-well culture plates. The NG
domain was crystallized with the his tag present at
the C terminus. Crystals were obtained for both
protein samples at 4°C. Crystals of the full-length
protein were obtained in 100 mM NaCitrate, 50 mM
bis-Tris pH 6.5 and 15% isopropanol. The crystals of
the NG domain grew in 50 mM bis-Tris pH 6.0, 200
mM Li2SO4,and 18% PEG 8000 at 4°C within 1 week
to a maximum size of around 0.3 3 0.4 3 3.0 mm.
These crystals were suitable for x-ray diffraction.
Data Collection and Reduction
Initially, inspection of the crystals of the fulllength protein, the NG domain, as well as heavy
metal soaks were done at room temperature on an
MAR research image plate on a rotating anode. For
data collection, crystals of the NG domain were
mounted in loops and flash-frozen with liquid nitrogen to 100 K with an Oxford Cryosystems cryostream. Before freezing the crystals were soaked in a
cryobuffer containing 50 mM bis-Tris pH 6.0, 18%
PEG 8000, and 10% PEG 400, for 2 minutes. Native
data were collected using BL-19 at the European
Synchrotron Radiation Facility (ESRF) in Grenoble.
Data were collected with a detector-to-crystal distance of 14 cm (180-mm diameter plate, l 5 1.0 Å) in
1-in. frames and with a 2-minute exposure time. All
data have been integrated with Denzo and Scalepack. 17
Results and Discussion
Using the expression protocol described above,
full-length FtsY and the NG domain can routinely be
produced in E. coli with yields ranging from 10 to 30
mg/l. The Se-Met containing NG domain is slightly
less well expressed and yields about 7 mg/l. The
287
DOCKING PROTEIN FTSY OF E. COLI
TABLE I. Data Statistics
Total
observations/
unique
Complete- Rsym†
dmin*
reflections
ness (%)
(Å)
(%)
Crystal
NG-FtsY in
house
NG-FtsY BL19
Se-NG-FtsY
BL19
Fig. 1. a: Needle-like crystals of the full-length FtsY protein. b:
Monoclinic crystals of the NG-domain of FtsY (with C-terminal his
tag). The approximate size is 0.2 3 0.2 3 0.3 mm.
incorporation of Se-Met was tested by mass spectrometry showing that all six methionines have been
replaced by Se-Met. The exchange of GDP versus
GMP-PNP seems to be crucial for obtaining well
ordered crystals—even when the nucleotide is no
longer present in the protein used for crystallization,
as determined by HPLC analysis. Without following
the exchange protocol or using cocrystallization, only
small bundles of needles were obtained. So far, we
have no explanation for this puzzling observation.
One possibility is that an impurity is removed by the
additional gel filtration step after the nucleotide
exchange. However, including this step in the purification without following the exchange protocol did
not have the same effect. It seems more likely that
the exchange is needed to get the protein in one
conformation.
Needle-like crystals were obtained for the fulllength protein (Fig. 1a), but the size was not sufficient to allow crystallographic experiments. Further
attempts to improve the quality of these crystals
were unsuccessful so far. Most likely, growth in the
third dimension is hampered by the acidic Nterminal domain. The existence of a stable proteolytic fragment of FtsY containing only the NG domain18 suggested the use of this fragment for
crystallographic studies. Monoclinic crystals of the
NG domain were obtained, see Figure 1b. The crystals belong to the space group P21 with unit cell
dimensions a 5 32.2, b 5 79.57, c 5 59.21, and b
angle 5 94.45. Assuming one protein molecule per
asymmetric unit, the crystal volume per protein
mass (Vm) is 2.3 Å3/Da, corresponding to a solvent
content of 37%. This is within the range commonly
observed for protein crystals.19
The crystals diffract beyond 3 Å at room temperature, but radiation damage decreased the diffracton
limit to 4 Å after 7 to 9 hours. Cryocooling effectively
eliminated radiation damage and also increased the
resolution to about 2.5 Å. Cryocooling caused changes
in the b angle and in the b axis of the crystals. A
native dataset at 110 K was collected by using
synchrotron radiation at the ESRF in Grenoble
2.5
1.8
52959/10181
190204/24107
97.2
88.2
3.0
6.9
2.7
45999/7468
89.1
9.0
*Minimum Bragg spacing with (I )/s at least 2.5.
†R
sym 5 S 0 Iobs 2 Iavg 0 /S Iavg .
BL-19. Synchrotron radiation increased the averaged resolution to beyond 1.8 Å. For the statistics of
the different native data sets see Table I.
Heavy metal soaks are in progress—so far without
success due to problems caused by nonisomorphism
of the soaked crystals with the native ones. Since the
NG domain contains six methionine residues it
seems possible to use the MAD approach for solving
the structure.20 Crystals of the Se-Met-containing
NG domain have been obtained following the same
protocol as for the ‘‘native’’ protein. The Se-Met
crystals are isomorphous with the ‘‘native’’ crystals and
diffract to about 2.8 Å on a rotating anode (Table I).
ACKNOWLEDGMENTS
We thank Andy Thompson from the EMBLoutstation Grenoble for help with data collection and
the ESRF for access to BL-19.
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