Aumelas, André

Calas, Bernard

Chavanieu, Alain

Dorsselaer, Alain,

Dumas, Christian

Ganz, Tomas

Hoh, Francois

Lehrer, Robert

Sanchez, Jean Frédéric

Strub, Jean,

Strub, Marie-Paule

English

Archive Ouverte en Sciences de l'Information et de la Communication

HAL Id: hal-02359559
https://hal.archives-ouvertes.fr/hal-02359559
Submitted on 13 Nov 2019
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
Expression, purification, crystallization and preliminary
X-ray analysis of the cathelicidin motif of the
protegrin-3 precursor
Jean Frédéric Sanchez, Francois Hoh, Marie-Paule Strub, Jean Strub, Alain
Dorsselaer, Robert Lehrer, Tomas Ganz, Alain Chavanieu, Bernard Calas,
Christian Dumas, et al.
To cite this version:
Jean Frédéric Sanchez, Francois Hoh, Marie-Paule Strub, Jean Strub, Alain Dorsselaer, et al.. Ex-
pression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the
protegrin-3 precursor. Acta Crystallographica Section D: Biological Crystallography, International
Union of Crystallography, 2001, 57, pp.1677 - 1679. ￿hal-02359559￿
Acta Cryst. (2001). D57, 1677±1679 Sanchez et al.  Cathelicidin motif of the protegrin-3 precursor 1677
crystallization papers
Acta Crystallographica Section D
Biological
Crystallography
ISSN 0907-4449
Expression, purification, crystallization and
preliminary X-ray analysis of the cathelicidin motif
of the protegrin-3 precursor
Jean FreÂdeÂric Sanchez,a FrancËois
Hoh,a Marie-Paule Strub,a
Jean Marc Strub,b Alain Van
Dorsselaer,b Robert Lehrer,c
Tomas Ganz,c Alain Chavanieu,a
Bernard Calas,a Christian
Dumasa and AndreÂ Aumelasa*
aCentre de Biochimie Structurale, UMR 5048
CNRS-UM1/UMR 554 INSERM-UM1,
UniversiteÂ Montpellier 1, FaculteÂ de Pharmacie,
15 Avenue Charles Flahault, 34060 Montpellier
CEDEX 2, France, bLaboratoire de SpectromeÂtrie
de Masse Bio-Organique, 1 Rue Blaise Pascal,
FaculteÂ de Chimie, 67000 Strasbourg, France,
and cDepartment of Medicine, Center for the
Health Sciences, Los Angeles, CA 90024, USA
Correspondence e-mail:
aumelas@cbs.univ-montp1.fr
# 2001 International Union of Crystallography
Printed in Denmark ± all rights reserved
Numerous precursors of antibacterial peptides with unrelated
sequences share a similar prosequence which belongs to the
cathelicidin family of proteins. The three-dimensional structure of
this cathelicidin motif, which contains two disul®de bonds, has not yet
been reported. The cathelicidin motif (ProS) of the protegrin-3
precursor was overexpressed in Escherichia coli as a His-tagged
protein. The His6 tag was removed by thrombin cleavage. ProS was
puri®ed to homogeneity and single crystals were obtained by the
hanging-drop vapour-diffusion method at pH 3±4. Preliminary X-ray
diffraction analysis indicated that these crystals belong to the
hexagonal space group P6122 or P6522, with unit-cell parameters
a = b = 51.42, c = 134.25 AÊ . These crystals diffracted beyond 2.75 AÊ
(1.9 AÊ at ESRF) and contain one molecule per asymmetric unit.
Received 10 May 2001
Accepted 24 July 2001
1. Introduction
Numerous precursors of antibacterial peptides
with unrelated sequences, including the
protegrin precursors, share a similar pro-
sequence of 96±101 residues and belong to
cathelicidin family (Gennaro et al., 1998;
Gennaro & Zanetti, 2000; Ritonja et al., 1989;
Scocchi et al., 1997; Storici et al., 1996; Wang et
al., 1998; Zanetti et al., 1995, 2000; Zhao et al.,
1994). Protegrins (PG-1 to PG-5, 16±18 resi-
dues) are a family of ®ve antibacterial peptides
isolated from porcine leucocytes (Kokryakov
et al., 1993). They are initially synthesized as a
149-residue precursor (147 residues for PG-2)
devoid of antibacterial activity, in which three
regions have been identi®ed: the signal peptide
(sequence 1±29), the prosequence (sequence
30±130) and the protegrin sequence (sequence
131±148 for PG-1 and PG-3). The Gly149 is
removed in a well known amidation step
(Merkler, 1994; Suzuki et al., 1990; Zhao et al.,
1997). The protegrin prosequence, referred to
as ProS, contains four cysteines engaged in two
disul®de bonds. Their 1±2, 3±4 arrangement
was clearly established for probactenecin-7, a
bovine cathelicidin (Storici et al., 1996). Taking
into account the high degree of sequence
identity between the probactenecin and ProS
sequences (74±78%) and the alignment of the
four cysteines, the disul®de-bond arrangement
of ProS was assumed to be identical to that of
probactenecin. Therefore, the two disul®de
bonds of ProS are Cys85±Cys96 and Cys107±
Cys124. The detailed three-dimensional struc-
ture of this widespread cathelicidin motif is not
yet known. To determine the structure of the
cathelicidin motif of the protegrin-3 precursor,
ProS was overexpressed in E. coli. Here, we
report the overexpression, puri®cation, crys-
tallization and preliminary X-ray diffraction
analysis of the recombinant ProS.
2. Materials and methods
2.1. Overexpression and purification of the
His-tagged ProS
A PCR product containing the coding
sequence of ProS was generated from the
plasmid pBluescript including the pro-
protegrin-3 cDNA (Panyutich et al., 1997). The
cDNA encoding for the ProS sequence
(residues 30±130) was subcloned into the
NdeI/BamHI sites of the pET-15b plasmid
vector (Novagen) leading to the N-terminus in
frame fusion with the His tag. E. coli strain
BL21(DE3), transformed with the recombi-
nant plasmid, was grown in 400 ml of medium
(20 g lÿ1 tryptone, 10 g lÿ1 yeast extract, 5 g lÿ1
sodium citrate, 5 g lÿ1 KH2PO4 adjusted to pH
7.0) containing 100 mg lÿ1 ampicillin. After
overnight growth at 310 K, the culture was
used to inoculate 4 l of the same medium
supplemented with ampicillin (100 mg lÿ1),
MgSO4 (10 mM), glucose (5 g l
ÿ1), biotin
(1 mg lÿ1), thiamine (10 mg lÿ1) and nicotin-
amide (10 mg lÿ1) and grown at 310 K for 3 h.
Expression was induced for 4 h by addition of
1 mM IPTG. The cells were harvested by
centrifugation for 15 min at 8000 rev minÿ1.
Cells, 20 g wet weight, were resuspended in
50 ml of ice-cold buffer A (100 mM Tris±HCl
pH 7.5, 150 mM NaCl) containing 5 mM
benzamidine. The mixture was homogenized
and freezed/thawed three times. The lysate was
1678 Sanchez et al.  Cathelicidin motif of the protegrin-3 precursor Acta Cryst. (2001). D57, 1677±1679
crystallization papers
kept on ice and probe-sonicated for 1 min
with 0.1 s bursts at 340 W. Particulate
material was then removed by centrifugation
at 20 000g for 30 min at 277 K. The super-
natant was further clari®ed by centrifugation
at 90 000g for 30 min and applied to a Q-
Sepharose (Amersham Pharmacia Biotech)
column (20  2.6 cm) equilibrated with
buffer A.
The ¯owthrough containing the protein
was applied to an Ni-NTA Super¯ow
(Qiagen) column (2  2.5 cm) equilibrated
with buffer A. The column was washed with
30 ml of buffer A, 50 ml of buffer A
containing 1 M NaCl and then re-equili-
brated with buffer A. The protein was eluted
with 0.5 M imidazole in the above buffer and
10 ml fractions of eluent were analyzed by
Coomassie-staining SDS±PAGE gels. The
protein-containing fractions 1±4 were
pooled and concentrated 20 times using an
Ultrafree (Millipore) ®lter with a MW cutoff
of 5000 Da. Imidazole was removed by
several concentration/dilution cycles with
buffer A. The concentration of the sample
was estimated by UV±visible spectroscopy
using a calculated extinction coef®cient of
2800 molÿ1 cmÿ1 at 280 nm.
2.2. His-tag cleavage and purification of
ProS
The protein was digested at 298 K in the
presence of 2 mM CaCl2 with 10 units of
thrombin per milligram of protein. The
cleavage was complete after 1 h. The protein
was puri®ed by gel ®ltration on a Sephadex
HR-100 (Pharmacia Biotech) column (100
1.6 cm) equilibrated with buffer B (50 mM
sodium phosphate, 50 mM NaCl pH 7.0).
1 ml fractions were collected at a ¯ow rate of
0.6 ml minÿ1. The protein was eluted as a
single peak and the corresponding fractions
pooled and concentrated. About 10±12 mg
of puri®ed ProS were obtained from 20 g
wet weight of cells. Purity was controlled by
Coomassie-staining SDS±PAGE gels, 1H
NMR and mass spectrometry (Fig. 1).
2.3. Crystallization
The hanging-drop vapour-diffusion
method was used to screen and to optimize
the crystallization conditions. The drops
contained equal volumes (1 ml) of ProS
protein solution (35 mg mlÿ1 in 10 mM Tris±
HCl buffer pH 7.0) and reservoir solution.
The optimized reservoir solution consisted
of 1.2±1.4 M ammonium sulfate and 0.1 M
sodium citrate buffer at pH 3.8. Two types of
hexagonal diffraction-quality crystals were
reproducibly obtained within a week at
291 K. From drops containing 1.2 M
ammonium sulfate reservoir solution,
crystals reached dimensions of
about 0.2  0.1  0.05 mm, whereas
larger crystals were obtained from
drops containing 1.4 M ammonium
sulfate reservoir solution (Fig. 2).
Prior to data collection, crystals
were transferred to cryoprotectant
mother liquor supplemented with
30% glycerol and ¯ash-frozen.
3. Results and discussion
Well diffracting crystals of the
recombinant ProS protein, corre-
sponding to the cathelicidin prote-
grin-3 precursor, were obtained.
X-ray data were collected at 100 K
on a MAR Research image-plate
detector (Hamburg, Germany)
mounted on a Rigaku RU-200
rotating-anode generator (Tokyo,
Japan) operating at 40 kV and
90 mA. The crystal-to-detector distance was
140 mm and the oscillation range was 0.5
per ®lm (25 min exposure time). The images
were processed and scaled with the HKL
suite of programs (Otwinowski & Minor,
1997). The autoindexing procedure indi-
cated that the two types of crystals belong to
a primitive hexagonal space group, with
unit-cell parameters a = b = 51.42,
c = 134.25 AÊ . The lattice was assigned to one
of the enantiomorphic space groups P6122
or P6522. Considering a molecular weight of
11 718 Da for the protein, the Matthews
coef®cient was determined to be
2.19 AÊ 3 Daÿ1, assuming one molecule per
asymmetric unit, with a corresponding
solvent content of 44%. Table 1 summarizes
the data-collection statistics. While good-
quality data could be collected to 2.75 AÊ
resolution on a conventional X-ray source,
the same crystal diffracted beyond 1.9 AÊ
resolution using synchrotron radiation
(ESRF, Grenoble, FIP beamline).
Initial attempts to solve the structure by
molecular replacement using various struc-
tures supposed to share a similar global fold
(PDB entries 1cew, 1stf, 1dvd, 1mnl, 1eqk,
1g96) as search models have so far proven
unsuccessful. Screening for heavy-atom
derivatives is now under way.
Figure 1
Molecular mass spectrum of the recombinant ProS. Mass
analysis of the recombinant ProS was performed using the
electrospray mass spectrometry technique (ES-MS) on a VG
Bio-Q quadrupole with a mass range of 4000 Da (Bio-Tech,
Manchester, England) in the positive mode. The protein was
desalted on Zip-Tip (Millipore) and 10 pmol were used for
mass analysis. The calibration was performed using the multiply
charged ions produced by a separate introduction of horse
heart myoglobin (16 951.4 Da).
Figure 2
Photomicrographs of the two types of crystals
obtained from drops containing (a) 1.2 M and (b)
1.4 M ammonium sulfate of the recombinant ProS
overexpressed in E. coli. Typical dimensions are
about 0.1  0.1  0.08 mm and 0.2  0.05  0.05 mm
for A and B, respectively.
Table 1
Data-collection statistics.
Values in parentheses refer to data in the last resolution
shell (2.80±2.75 AÊ ).
Space group P6122 or P6522
Unit-cell parameters (AÊ )
a = b 51.42
c 134.25
Resolution range (AÊ ) 23±2.75
Asymmetric unit content Monomer
Total No. of re¯ections 21019
No. of unique re¯ections 3110
Redundancy 6.6
Average I/(I) 16.1 (3.6)
Completeness (%) 91.3 (58)
Rmerge² (%) 6.2 (28.6)
² Rmerge =
P jIh ÿ hIhij=P Ih .
Acta Cryst. (2001). D57, 1677±1679 Sanchez et al.  Cathelicidin motif of the protegrin-3 precursor 1679
crystallization papers
This research was supported by a grant from
the MinisteÁ re de l'Education Nationale, de
la Recherche et de la Technologie (JFS). We
acknowledge the opportunity to test preli-
minary diffraction data at the ESRF
synchrotron (Grenoble, France) and are
indebted to Dr S. L. Salhi for editorial
revision of the manuscript.
References
Gennaro, R., Scocchi, M., Merluzzi, L. & Zanetti,
M. (1998). Biochim. Biophys. Acta, 1425, 361±
368.
Gennaro, R. & Zanetti, M. (2000). Biopolymers,
55, 31±49.
Kokryakov, V. N., Harwig, S. S., Panyutich, E. A.,
Shevchenko, A. A., Aleshina, G. M., Shamova,
O. V., Korneva, H. A. & Lehrer, R. I. (1993).
FEBS Lett. 327, 231±236.
Merkler, D. J. (1994). Enzyme Microb. Technol.
16, 450±456.
Otwinowski, Z. & Minor, W. (1997). Methods
Enzymol. 276, 307±326.
Panyutich, A., Shi, J., Boutz, P. L., Zhao, C. &
Ganz, T. (1997). Infect. Immun. 65, 978±985.
Ritonja, A., Kopitar, M., Jerala, R. & Turk, V.
(1989). FEBS Lett. 255, 211±214.
Scocchi, M., Wang, S. & Zanetti, M. (1997). FEBS
Lett. 417, 311±315.
Storici, P., Tossi, A., Lenarcic, B. & Romeo, D.
(1996). Eur. J. Biochem. 238, 769±776.
Suzuki, K., Shimoi, H., Iwasaki, Y., Kawahara, T.,
Matsuura, Y. & Nishikawa, Y. (1990). EMBO J.
9, 4259±4265.
Wang, Y., Agerberth, B. & Johansson, J. (1998). J.
Protein Chem. 17, 522±523.
Zanetti, M., Gennaro, R. & Romeo, D. (1995).
FEBS Lett. 374, 1±5.
Zanetti, M., Gennaro, R., Scocchi, M. & Skerlavaj,
B. (2000). Adv. Exp. Med. Biol. 479,
203±218.
Zhao, C., Liaw, L., Lee, I. H. & Lehrer, R. I.
(1997). FEBS Lett. 410, 490±492.
Zhao, C., Liu, L. & Lehrer, R. I. (1994). FEBS
Lett. 346, 285±288.


Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor

https://hal.archives-ouvertes.fr/hal-02359559/document

Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor

Abstract

Similar works

Full text

Available Versions

Archive Ouverte en Sciences de l'Information et de la Communication