Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 angstrom using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2(1) (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 angstrom, beta = 102.97 degrees) and the orthorhombic space group P2(1)2(1)2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 angstrom), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.62875075

Barbosa, JARG

Cancado, FC

Ferreira, C

Marana, SR

Terra, WR

Valerio, AA

Acta Crystallographica Section F Structural Biology and Crystallization Communications

Repositorio da Producao Cientifica e Intelectual da Unicamp

crystallization communications
750 doi:10.1107/S1744309106024201 Acta Cryst. (2006). F62, 750–752
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Crystallization, data collection and phasing of two
digestive lysozymes from Musca domestica
S. R. Marana,a‡ F. C. Cançado,a‡
A. A. Valério,b,c C. Ferreira,a
W. R. Terraa and
J. A. R. G. Barbosab,c,d*
aDepartamento de Bioquı́mica, Instituto de
Quı́mica, Universidade de São Paulo, São
Paulo, Brazil, bCentro de Biologia Molecular e
Estrutural (CeBiMe), Laboratório Nacional de
Luz Sı́ncrotron (LNLS), CP 6192, Campinas,
SP 13084-971, Brazil, cDepartamento de
Bioquı́mica, Universidade Estadual de
Campinas, Campinas, Brazil, and
dDepartamento de Genética e Evolução,
Universidade Estadual de Campinas,
Campinas, Brazil
‡ These authors contributed equally to this
work.
Correspondence e-mail: joao@lnls.br
Received 4 April 2006
Accepted 23 June 2006
Lysozymes are mostly known for their defensive role against bacteria, but in
several animals lysozymes have a digestive function. Here, the initial crystallo-
graphic characterization of two digestive lysozymes from Musca domestica are
presented. The proteins were crystallized using the sitting-drop vapour-diffusion
method in the presence of ammonium sulfate or PEG/2-propanol as the
precipitant. X-ray diffraction data were collected to a maximum resolution of
1.9 Å using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the
monoclinic space group P21 (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å,
 = 102.97) and the orthorhombic space group P21212 (unit-cell parameters
a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved
by molecular replacement and structure refinement is in progress.
1. Introduction
Lysozymes (EC 3.2.1.17) catalyze the hydrolysis of 1,4--linkages
between N-acetylmuramic acid and N-acetylglucosamine residues in
the peptidoglycan of the bacteria cell wall. Based on sequence and
structure similarities, these enzymes are classified into families 22, 23,
24, 25 and 73 of the glycoside hydrolases (Coutinho & Henrissat,
1999).
Family 22 contains 219 type C lysozymes from vertebrates
(amphibians, fishes, birds and mammals), invertebrates (crustaceans,
arachnids and insects) and fungi (Coutinho & Henrissat, 1999). Hen
egg-white lysozyme (HEWL) is probably the best characterized
member of this family.
Lysozymes from family 22 are usually part of the defence system
against bacteria (Jollès & Jollès, 1984). However, some of these
lysozymes are also involved in the digestion of bacteria in vertebrates
that have a fermentation chamber in their foregut (for example,
ruminants such as Bos taurus and primates such as Presbytis entellus)
and in insects that feed on decomposing organic material (for
example, the housefly Musca domestica; Lemos & Terra, 1991; Prager,
1996). These lysozymes present several adaptations to digestive
function: a high expression level in the gut, a resistance to proteinase
hydrolysis and bacteriolytic activity with an acidic pH optimum
(Dobson et al., 1984). Sequence alignments have been used to
propose the molecular basis of these adaptations (Prager, 1996; Regel
et al., 1998), but these hypotheses still remain to be tested.
The tertiary structure of digestive lysozymes is not known,
although 15 different lysozymes from family 22 have already been
crystallized, corresponding to 452 structures in the PDB. Despite the
fact that all lysozymes from family 22 share the same fold, slight
differences in their structures may be correlated to adaptations of
lysozyme to the digestive function.
Two digestive lysozymes are found in the M. domestica midgut
(lysozyme 1, AAQ20048; lysozyme 2, AAQ20047). Lysozyme 1 (122
amino acids, 13 816 Da) has previously been submitted to biochem-
ical characterization, showing that the lytic activity of this lysozyme
has a pH optimum of 4.5 and its affinity for bacterial cell walls
decreases as the ionic strength of the medium becomes higher
(Lemos et al., 1993; Ito et al., 1995). Lysozyme 2 (122 amino acids;
13 890 Da) is very similar to lysozyme 1 (81% similarity; 70% iden-
tity), but still remains to be characterized.
# 2006 International Union of Crystallography
All rights reserved
In this paper, the crystallization of lysozymes 1 and 2 is reported.
The tertiary structure of these digestive lysozymes may contribute to
the comprehension of the molecular basis of the adaptation of these
lysozymes to digestive function.
2. Methods
2.1. Crystallization
Both lysozymes were expressed as recombinant protein in Pichia
pastoris. An initial step of purification was ammonium sulfate
precipitation followed by ion-exchange chromatography (Cançado et
al., 2006). Samples of purified lysozyme 1 (9.6 mg ml1) and lysozyme
2 (9.8 mg ml1) from M. domestica were submitted to crystallization
trials using the sitting-drop vapour-diffusion method. Both samples
were in 10 mM 2-morpholinoethanesulfonic acid (MES) pH 5.5
buffer. The experiment was set up by mixing equal volumes (1 ml) of
protein and reservoir solution in Cryschem plates. Initial screening
was performed using the commercial kits Crystal Screen and Crystal
Screen II from Hampton Research according to the manufacturer’s
instructions. Conditions showing crystalline structures were refined
by varying the pH of the buffers, the concentrations of precipitants
and the drop volumes to yield suitable crystals for X-ray data
collection. In the case of lysozyme 2, the condition was further refined
with additive solutions in two ways: (i) by using the Additive Screen
from Hampton Research according to the manufacturer’s instruc-
tions and (ii) by mixing the solutions of the Crystal Screen from
Hampton Research with the crystallization drop (Birtley & Curry,
2005). In the latter case, 0.2 ml of the Crystal Screen was mixed with
1.5 ml of protein solution and 1.3 ml of the reservoir solution, leading
to a total drop volume of 3 ml. All experiments were carried out at
291 K.
2.2. Data collection, processing and phasing
Crystallographic data were collected at the protein crystallography
beamline D03B-MX1 at Laboratório Nacional de Luz Sı́ncroton
(LNLS), Campinas, Brazil. This beamline is equipped with a MAR
CCD detector with a circular X-ray-sensitive surface of 165 mm in
diameter combined with a MAR DTB goniostat. Crystals were
scooped straight from the drop and cooled directly in a nitrogen-gas
stream to 100 K in order to minimize radiation damage to the crystals.
Data collection was carried out using the oscillation method with a
1.0 oscillation per frame and radiation of wavelength around 1.43 Å.
D03B-MX1 is a monochromatic beamline and operates at this
wavelength as a compromise between flux and absorption. The
solution in which the crystals were grown provided partial protection
against ice formation for lysozyme 1. A solution containing 20%
glycerol, 9% polyethyleneglycol (PEG) 4000, 12% 2-propanol and
0.05 M sodium citrate pH 4.2 was used as a cryoprotectant in the case
of lysozyme 2. The crystals were soaked for 5–10 s in this solution
prior to flash-cooling in nitrogen gas. The data set was processed
using HKL-2000 (Otwinowski & Minor, 1997) and the CCP4 package
(Collaborative Computational Project, Number 4, 1994). Molecular
replacement was performed with the MOLREP program (Colla-
borative Computational Project, Number 4, 1994; Vagin & Teplyakov,
1997) using one monomer of the lysozyme structure from hen egg
white deposited in the PDB with code 1hew (Cheetham et al., 1992)
depleted of its waters and ligand as a search model. The identity of
the 1hew sequence with lysozymes 1 and 2 is 38.5 and 36.2%, while
the similarity is 55.4 and 53.8%, respectively. In the case of lysozyme
2, molecular replacement was performed with the partially refined
lysozyme 1 as a search model.
3. Results and discussion
Initial crystallization trials with lysozyme 1 and lysozyme 2 resulted in
microcrystals in some conditions. In order to improve the quality of
these crystals, refinement of these conditions was performed. Crystals
grew in one to two weeks, leading to crystals of between 50 and
1000 mm in the longest dimension. Crystals usually formed clusters of
plates, especially for lysozyme 2. To minimize this clustering in the
crystallization of lysozyme 2, additives were tested.
The best condition found for lysozyme 1 was 1.4 M ammonium
sulfate, 0.1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
sodium salt (sodium HEPES) pH 7.5 and 1% PEG 400 (Fig. 1). The
best condition for crystals of lysozyme 2 was the drop mixture
described in x2, where the reservoir solution consisted of 28%
crystallization communications
Acta Cryst. (2006). F62, 750–752 Marana et al.  Digestive lysozymes 751
Figure 1
(a) Crystals of lysozyme 1 measuring approximately 1000 mm in the longest
dimension.
Figure 2
(a) Crystals of lysozyme 2 measuring approximately 200 mm in the longest
dimension.
2-propanol, 0.115 M sodium citrate pH 4.2 and 21% PEG 4000 and
the additive solution was No. 26 of Hampton Research Crystal Screen
(0.2 M ammonium acetate, 0.1 M sodium citrate pH 5.6 and 30%
2-methyl-2,4-pentanediol) (Fig. 2). The cluster shown in Fig. 2 is not a
single crystal. In order to avoid multiple diffraction patterns, we
needed to break the cluster by touching its vertex, thus separating the
plates, which could be used as single crystals.
The crystals of both lysozymes 1 and 2 yielded diffraction data that
were processed to a maximum resolution of 1.9 Å. The rotation
method was used for data collection and no prediction of the best
strategy (starting angle) was made for the data collection. Space
group P21 was assigned for lysozyme 1, while the crystal of lysozyme 2
showed the symmetry and systematic absences of the orthorhombic
space group P21212. Table 1 summarizes the data-collection statistics.
The Matthews coefficient (Matthews, 1968) for two molecules of
lysozyme 1 in the asymmetric unit was calculated to be 2.3 Å3 Da1,
giving a solvent content of 45.7%. For two molecules of lysozyme 2 in
the asymmetric unit, the coefficient was 2.1 Å3 Da1, with a solvent
content of 40.4%. Clear peaks in the rotation and translation func-
tions were observed for the molecular-replacement solutions of
lysozyme 1. The initial electron-density maps of the proteins clearly
showed the expected differences in the side chains between 1hew and
lysozyme 1. After a few cycles of refinement, the model of lysozyme 1
was used as a search model for lysozyme 2. Once again, clear peaks in
the rotation and translation functions were found and the electron-
density maps showed the differences between lysozyme 1 and 2. The
crystallographic models of these proteins are being built and refined.
This work was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP grants 04/02017-9, 04/02225-0, 02/
10634-2, 01/08198-7, 01/07531-4 and 00/10266-8; SMolBNet),
Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico
(CNPq) and Associação Brasileira de Tecnologia de Luz Sı́ncrotron
(ABTLuS). We also thankfully acknowledge the support from Dr
Rogério Meneghini and the technical assistance of Andréia N. Meza.
References
Birtley, J. R. & Curry, S. (2005). Acta Cryst. D61, 646–650.
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613–628.
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crystallization communications
752 Marana et al.  Digestive lysozymes Acta Cryst. (2006). F62, 750–752
Table 1
Data-collection and processing statistics.
Values in parentheses are for the highest resolution shell.
Lysozyme 1 Lysozyme 2
Space group P21 P21212
Unit-cell parameters (Å, ) a = 36.52, b = 79.44,
c = 45.20,
 =  = 90.00,
 = 102.97
a = 73.90, b = 96.40,
c = 33.27,
 =  =  = 90.00
Mosaicity () 0.7 0.8
Temperature (K) 100 100
Wavelength (Å) 1.431 1.427
Oscillation () 1.0 1.0
Cystal-to-dectector distance (mm) 80.0 80.0
No. of frames 171 127
Resolution limits (Å) 40.00–1.9 (1.97–1.90) 30.00–1.9 (1.97–1.90)
I/(I) after merging 23.8 (5.6) 14.4 (2.1)
Completeness (%) 100.0 (99.8) 90.7 (74.1)
Multiplicity 3.5 (3.4) 3.9 (1.8)
Rsym 0.052 (0.222) 0.089 (0.304)
No. of reflections 70004 69179
No. of unique reflections 19857 (1953) 17698 (1423)
B factor (Wilson plot) (Å2) 20.0 23.3


Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica

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Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica

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