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PROTEINS: Structure, Function, and Genetics 25:112-119 (1996)
Purification, Stabilization, and Crystallization of a
Modular Protein: Grb2
J.P. Guilloteau,' N. Fromage,' M. Ries-Kautt? S. Reboul,' D. Bocquet,' H. Dubois,' D. Faucher?
C. Colonna,' A. Ducruix? and J. Becquart'
'Service de Biochimie, RhGne-PoulencRorer SA, 94403 VitrylSeine, France; 'Laboratoire de Bwlogie Structurale,
CNRS, 91198 GiflYuette, France; 3Seruice de Microbiologie MolCculaire, RhGne-Poulenc Gencell, 94403
VitrylSeine,France
ABSTRACT
We report here the purification and the crystallization of the modular
protein Grb2. The protein was expressed as a
fusion with glutathione-S-transferaseand purified by affinity chromatography on glutathione
agarose. It was apparent from reverse phase
chromatography that the purified protein was
conformationallyunstable.Instability was overcome by the addition of 100 mM arginine to
the buffers. Because Grb2 appeared to be extremely sensitive to oxidation, crystallization
experiments were performed with a dialysis
button technique involving daily addition of
fresh DTT to the reservoirs. The presence of 8 to
14% glycerol was necessary to obtain monocrystals. These results are discussed in relation
with the modular nature of Grb2.
0 1996 WiIey-Liss, Inc.
Key words: Grb2, modular proteins, purification, crystallization, stabilizing
agents, arginine, DTT, glycerol
INTRODUCTION
Many large proteins are combinations of several,
clearly identifiable, autonomously folding modules.*
They have recently been the object of tentative classification~.~
These
- ~ modular proteins play various
fundamental biological roles, including cell adhesion, clotting, fibrinolysis, and signal transduction.
Despite the considerable interest in their tertiary
structure, very few structures of intact modular proteins, containing more than two modules, have been
reported so far (a monoclonal antibody6 and the protein Grb27).The three-dimensional structural determination of modular proteins is hampered by the
difficulty of crystallizing such protein^.^ We present
here the strategy that we used for the crystallization
of Grb2. Some steps are extensively detailed and are
given as experimental elements that may document
the crystallization of modular proteins and could be
of interest for other projects.
*Modules are autonomously folded homologous structures.
They are defined by consensus sequences of 40-100 conserved
residues, encoded on discrete exons and bordered by introns of
identical phase.','
0 1996 WILEY-LISS, INC.
Grb2 (Growth factor Receptor Bound protein 2) is
a 25 kDa intracellular monomeric adaptor protein,'
made up of one Src homology 2 (SH2) module
flanked by two Src homology 3 (SH3) modules. SH2
and SH3 domains are protein modules (100 and 50
amino acids respectively) involved in protein-protein interactions. Grb2 plays a key role in the mitogenic signal transduction pathway linking tyrosine
kinase receptor to Ras a c t i ~ a t i o n . ~
Considerable work has been devoted to the study
of SH2 and SH3 modules. Numerous three-dimensional structures of isolated SH2 and SH3 modules
have been published. In particular, the structures of
the Grb2 N-terminal SH3I0-l3 and C-terminal
SH314 domains complexed with peptides were recently resolved. The regulatory domain of the Srcfamily tyrosine kinase Lck15 was the first published
structure with two juxtaposed SH2 and SH3 domains.
In addition to the interest due to its important role
in the mitogen signal transduction pathway, Grb2
constitutes a good model with which to study the
orientations of different SH2 and SH3 domains in an
entire protein.
EXPERIMENTAL PROCEDURES
Protein
Expression vector
Grb2 was expressed as a glutathione-s-transferase fusion protein, using the vector pGEX-TT (de-
Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoicacid);
DTT, D-L dithiothreitol; EDTA, ethylenediamine tetraacetic
acid; EGFR, epidermal growth factor receptor; FMOC, 9-fluorenyl-methoxycarbonyl; Grb2, growth factor receptor bound
protein 2; GSH, reduced glutathione; GST, glutathione
S-transferase; IRS-1, insulin receptor substrate 1; MPD,
Z-methyl-2,4-pentanediol;PDGFR, platelet-derived growth
factor receptor; PEG, polyethylene glycol; RPLC, reverse phase
liquid chromatography; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SH2(3), Src homology domain 2(3);TFA, trifluoroacetic acid.
Received September 26,1995; revision accepted November 3,
1995.
Address reprint requests to Jean-Pierre Guilloteau, RhBnePoulenc Rorer SA, CRVA, BLtiment Monod, Service de Biochimie, 13 quai J. Guesde, 94403 VitrylSeine, France.
Grb2 CRYSTALLIZATION
rived from pGEX-2T, Pharmacia, France) to ensure
a more efficient cleavage of the fusion by thrombin.
The particular feature of this vector lies in the sequence encoded between GST and the fusion partner
that contained two thrombin cleavage sites separated by a 9 amino acid linker.16 The complete Grb2
cDNA sequence was subcloned into this vector.
Expression
Transformed cells were grown in 7-liter fermentors under fed batch conditions at 37°C in a medium
composed of yeast extract, glucose, mineral salts,
oligoelements, and 100 pg/ml ampicillin. IsopropylP-D-thiogalactopyranoside was added to 1mM and 2
h later cells were recovered by centrifugation a t
10,OOOg for 10 min and frozen a t -80°C.
Purification
E . coli cells from 1liter of culture were disrupted
by homogenization with a minilab 6 (Rannie,
France) at a pressure of 1,000 bars at 4°C in one
volume of lysis buffer pH 7.3 (16 mM Na,HPO,, 5
mM EDTA, 150 mM NaC1, 1 mM Pefabloc), then
centrifuged at 10,OOOg for 20 min to remove insoluble cellular debris. GSH agarose gel (150 ml of gel
Sigma, France, G 4510) was suspended in the supernatant (crude extract) and gently agitated for 2h a t
4°C. The lysate was then removed by filtration on
glasfilter (por. 4).The agarose beads were washed
three times with 600 ml of lysis buffer. Grb2 was
then eluted by cleavage with thrombin (human
plasma thrombin, Sigma T3010). The agarose gel
was washed with one volume of thrombin cleavage
buffer (50 mM phosphate pH 8.0) for 15 min at room
temperature. After filtration on glasfilter, the agarose beads were suspended in two volumes of cleavage buffer to which 5 pg of thrombin per mg of fusion protein were added (the amount of fusionprotein bound to the gel was previously determined
with a 0.5 ml aliquot of gel). The gel was then gently
shaken at room temperature for 30 min. The eluate
was removed by filtration on glasfilter (por. 4). The
beads were washed twice with 1.5 bead volume of
cleavage buffer for 15 min, and the eluates were
pooled. The thrombin in the Grb2 eluate was removed through the addition of antithrombin I11 agarose (Sigma A 8293,l ml of gel for 100 pg of thrombin). After 45 min a t 4"C, the antithrombin gel was
removed by filtration on glasfilter (por. 4). Then DTT
was added to the Grb2 solution to a final concentration of 10 mM. The last purification step consisted of
Mono Q ion exchange chromatography (HR10/10).
The column was equilibrated with 20 mM phosphate
buffer pH 8.0, and the sample was loaded at 3 ml/min
and eluted with an NaCl gradient. The Grb2 monomer was eluted with 0.125 M NaCl and the dimer
with 0.22 M NaC1. To stabilize the protein, 1M arginine in Tris buffer pH 8.0 was added to a final concentration of 0.1 M and the solution was stored at 4°C.
113
Peptides
The following phosphotyrosine-containing peptides were obtained from Neosystem (Strasbourg,
France): L-10-S (EGFR Tyr1068: Leu-Pro-Val-ProGlu-Tyr(P03H2)-Ile-Asn-Gln-Ser),
G-8-F (IRS-1Tyr
895: Gly-Glu-Tyr(P03H2)-Val-Asn-Ile-Glu-Phe),
S11-E (PGDF Tyr 977: Ser-Val-Leu-Tyr(P03H2)-ThrAla-Val-Gln-Pro-Asn-Glu).
Proline-rich peptides (G-20-AhSOS-1: 1143-1162)
were synthesized in the solid phase on 0.1 mM [(hydroxymethyl) phenoxymethyll polystyrene resin by
using FMOC chemistry, on an Applied Biosystems
(USA) Model 431A peptide synthesizer. All the solvents and reagents used were purchased from Applied Biosystems. After synthesis, protecting groups
were removed and the peptides cleaved and precipitated by the addition of tert-butyl methyl ether.
Peptides were then purified by preparative HPLC on
a BioRad, France, RSL C18 100 A column, eluted
with an increasing linear acetonitrile gradient containing 0.07% TFA in water and lyophilized.
Protein-PeptideInteractions
Protein-peptide interactions were studied using a
BIAcore'" (Pharmacia, France, Biosensor) that allows quantitative analysis of molecular interactions
in real
The flow rate for all the experiments was 5 pl/min. Peptides were covalently bound
to a CM5 sensor chip (Pharmacia Biosensor) equilibrated in HBS buffer (10 mM Hepes, 150 mM NaCl,
5 mM EDTA, 0.005% Tween 20, pH 7.4) with the
following procedure: the carboxymethylated dextran
matrix was activated with 40 p1 of a mixture of
N-hydroxysuccinimide and N-ethyl-N'-(3-diethylaminopropy1)-carbodiimide (amine coupling kit,
Pharmacia Biosensor). Next, 40 pl of a 5 mg/ml peptide solution in 50 mM borate buffer pH 8.5 containing 1 M NaCl were injected. Following peptide injection, 35 p1 of 1 M ethanolamine were injected to
block unreacted activated groups. The immobilization of phosphotyrosine peptides was controlled with
a monoclonal antibody directed against phosphotyrosine (UBI, Lake Placid, NY). Interaction of Grb2
with immobilized peptides was performed with 20 p1
of a 10 pg/ml Grb2 solution in HBS buffer. After
Grb2 injection, 15 pl of 100 mM HC1 in water were
applied to the matrix to remove bound material and
to regenerate the matrix for the next injection.
Crystallization
Reagents
The following reagents were purchased from the
indicated sources: PEG (Fluka, France); MPD, sodium para-toluene-sulfonate (Merck, France, for
synthesis); sodium chloride, potassium thiocyanate
(Merck for analysis); arginine monohydrochloride
(Merck for biochemistry); sodium-potassium tartrate, glycerol (Prolabo, France, for analysis); so-
114
J.P. GUILLOTEAU ET AL.
dium citrate (Sigma molecular biology reagent); sodium acetate (Sigma ACS reagent); ammonium
sulfate (Sigma ultra); DTT (Sigma). MilliQ deionized water (Millipore, France) was used to prepare
the solutions.
Protein concentration
At the end of the purification process, the chromatographic fractions containing the monomeric
protein were collected. In these fractions, Grb2 was
a t a concentration between 0.1 and 1 mg/ml, in 20
mM pH 8.0 phosphate solution with 100 mM arginine, 10 mM DTT and 110-130 mM NaC1. Prior to
any crystallization experiment, the protein was simultaneously dialyzed and concentrated a t room
temperature with a MicroProdicon, Polyabo, France,
device equipped with a 2-liter reservoir and a 10
kDa cut-off dialysis membrane. The dialysis solution was a 50 mM pH 8.0 Tris/HCl solution containing 100 mM arginine and 5 mM DTT. The final protein concentration, ranging from 5 to 40 mg/ml, was
determined spectrophotometrically by measuring
the A,,,.
Crystallization
Stock solutions of all crystallizing agents were
prepared in 50 mM TridHCl, 100 mM arginine, pH
8.0 buffer. Further dilutions were performed with
the same buffer. For the dilution of the protein solutions, this buffer contained additionally 5 mM
DTT. The solutions were filtered with 0.22 pm cellulose acetate filter units.
The crystallization screening experiments were
achieved using the vapor diffusion method in Linbro
boxes, with 4 to 8 p1 hanging drops and 1ml reserv o i r ~ at
, ~4~and 19°C. Freshly concentrated DTT
was added to the reservoir in order to obtain a 5 mM
final concentration. The drops were prepared by
mixing equal volumes of protein and reservoir solutions, such that a two-fold increase in concentration
was expected for all components in the drop during
the course of the experiment.
Crystallizations by dialysis were performed in 10
pl microdialysis buttons with Spectrapor, France, 6
dialysis membranes (molecular weight cut-off =
6,000-8,000 Da). The buttons were soaked in 10 ml
reservoirs.
The crystallization experiments were stored at
19 ? 0.1"C in a thermal regulated chamber (Memmert, France) or a t 4 5 0.5"C in a refrigerator
(Brandt, France).
Space group determination
Space group and unit cell dimensions were determined from data collected with synchrotron radiation (A = 0.901 A), on the wiggler beam-line DW32
at LURE (Orsay, France),' by using a MarResearch,
Germany, Imaging plate.
RESULTS AND DISCUSSION
Purification
The GST-Grb2 fusion protein was expressed a t a
level of 10-15% of the total soluble protein in E. coli.
It was purified with high yield (200-600 mg/liter of
culture) to >95% homogeneity by affinity chromatography on GSH-agarose. After thrombin cleavage,
Grb2 is 80%pure, containing Grb2 fragments but no
GST. We finally obtained 30 mg pure (>98%) monomeric Grb2 after the polishing step on Mono Q (Fig.
1).The purification steps that appear to be critical
for the crystallization of Grb2 are detailed below.
Thrombin cleavage
To facilitate the cleavage between GST and Grb2,
we started from the construction described by Guan
and Dixonl' composed of one thrombin cleavage site
followed by a glycine-rich linker containing the sequence P-G-I-S-G-G-G-G-G.In order to avoid the addition of these nine amino acid residues to the N-terminal side of Grb2, we introduced another thrombin
cleavage site between this linker and Grb2. Experiments showed that the cleavages were sequential:
the thrombin site upstream to the linker being
cleaved first. So, an insufficient quantity of thrombin resulted in contamination of Grb2 with molecules of Grb2 plus linker. On the other hand, the
presence of two sites inside Grb2 susceptible to be
cleaved by thrombin (Ar8l-Gly2, and Arg17'Glyl") could lead to proteolysis. It was thus crucial
to optimize the thrombin concentration in order to
obtain a compromise between efficient cleavage
(with minor contamination by Grb2 plus linker) and
slight degradation of Grb2 (Fig. 2).
Elimination of thrombin
Concentrating Grb2 was a prerequisite for the
crystallization experiments. In this step remaining
traces of thrombin were detrimental. To remove residual thrombin after cleavage, we tested gels specific for thrombin adsorption. Antithrombin I11 agarose gel was preferable to paraminobenzamidine
agarose because of its better adsorption capacity, although a nonspecific adsorption of Grb2 onto the gel
was observed. In this way 98% of the thrombin was
removed. The remaining 2% contamination was
eliminated with ion exchange chromatography on
Mono Q. Electrophoresis of concentrated Grb2 solutions, that had been stored for several weeks, indicated that no degradation had occurred.
Elimination of GST and GST-Grb2
Weak desorption of GST-Grb2 from the GSH-agarose gel during the cleavage at pH 8.0 led to contamination by GST in subsequent steps. As Grb2
and GST have approximately the same molecular
weight, their separation could not be followed by
SDS-PAGE. Experiments of spiking GST and GST-
115
Grb2 CRYSTALLIZATION
Fig. 1. Coomassie blue-stained SDS-PAGE (14Y-Novex)
of the purification of Grb2 from E.
m/i.Lane 1: Molecular weight markers (kDa). Lane 2: Cells (10 pg). Lane 3: Total soluble protein.
Lane 4: Supernatant after binding onto GSH agarose. Lane 5: Purified GST-Grb2 (bead suspension) (2 pg). Lane 6: Grb2 after cleavage with thrombin (2 pg). Lane 7: Mono Q flow through. Lane
8: Grb2 monomer (0.125 M NaCI) (5 pg). Lane 9: Grb2 dimer (0.22 M NaCI) (5 pg).
Grb2 onto Mono Q allowed us to find the conditions
for which the two proteins were separated from
Grb2. A Western blot of the final Grb2 solution with
anti-GST polyclonal antibodies confirmed that GST
and GST-Grb2 had been eliminated.
Polishing step
A polishing step on Mono Q was introduced in the
purification process to obtain “crystallization grade
protein,” because contaminating molecules closely
related to Grb2 (fragments, Grb2 plus linker, oligomers, and GST-Grb2) could later interfere with the
crystal growth. GST and thrombin were eliminated
in the flow-through. The first major eluted peak corresponded to monomeric Grb2, and the second one
was identified as non-covalent dimeric Grb2. The
use of a smooth gradient allowed the separation of
Grb2 plus linker from Grb2, respectively in the rise
and fall of the first peak. It has to be noticed that
phosphate buffer, unusual for Mono Q , eliminated
Grb2 fragments better than Tris buffer did.
Characterization
The purity of the protein in the first peak was
checked by native and SDS-PAGE. Grb2 was characterized by Electrospray Mass Spectrometry (EMS)
and amino-terminal sequencing. Titration of the
sulfhydryl groups with DTNBZ1showed the absence
of a disulfide bond between the only two cysteines of
the protein (C3’ and C1’*). Size exclusion chromatography, analytical ultracentrifugation, and light
scattering experiments indicated that the concen-
trated solution was monodisperse and contained monomeric Grb2.
Because Grb2 cannot be tested enzymatically, the
binding activity of the protein was controlled using
BIAcore’”. Plasmon resonance with BIAcore’” was
ideally suited to analyzing the ability of the purified
protein to bind various peptides known to interact
with SH2 or SH3 domains. As described in the lite r a t ~ r e , 2we
~ ~observed
~~
specific interactions of
very high affinity between Grb2 and short phosphotyrosine-containing peptides derived from a major
insulin receptor substrate IRS-1, from the epidermal
growth factor receptor (EGFR), and from the platelet-derived growth factor (PDGFR). Grb2 was able to
bind L-10-S (EGFR) and G-8-F (IRS-1) either immobilized on the matrix or in solution. S-11-E (PDGFR)
was not recognized. In the same way, Grb2 had a
high affinity with the proline-rich peptide G-20-A.
Stability
Although Grb2 proved to be pure and monomeric
at the end of the purification process, the protein
stability i n solution vs. time had to be investigated
prior to any crystallization attempt. Verification
by electrophoresis, gel filtration chromatography,
and reverse phase liquid chromatography (RPLC)
showed:
- the formation of covalent dimers due to cysteine
oxidation,
- the formation of non-covalent dimers when the
solutions were stored deep-frozen, and
116
J.P. GUILLOTEAU ET AL.
Fig. 2. Coomassie blue-stained SDS-PAGE (14%N
-ovex)
of thrombin cleavage of the purified
fusion protein GST-Grb2. GST-Grb2 bound to glutathione-agarose beads was incubated with
various quantities of thrombin at room temperature for 90 min in cleavage buffer. The molecular
weight of GST-Grb2 and Grb2 is 50 kDa and 25 kDa, respectively. After thrombin cleavage,
fragments corresponding to internal sequences are around 18 kDa, 20-22 kDa and 3-4 kDa; Grb2
plus linker is around 26 kDa. The thrombin to substrate ratios (w/w) are as follows: Lane 1: 1/50.
Lane 2: 1/100. Lane 3: 1/200. Lane 4: 1/400. Lane 5: 1/800. Lane 6: 1/1600. The amino terminal
was sequenced to confirm the presence of Grb2 plus linker in lanes 4 and 5. Lane 7: Molecular
weights markers (kDa).
- the progressive appearance of conformational
heterogeneity, indicated by RPLC profiles,24 and
unrelated to oligomer formation.
The protein solution could be stabilized by:
- addition of 10 mM DTT to prevent from covalent
dimers,
- storage at 4°C to avoid the formation of non-covalent dimers, and
-addition of 100mM arginine as a stabilizing agent.
In the case of Grb2, reverse phase analysis appeared to be a valuable tool to detect the change of
protein conformation and to screen stabilizing
agents. Such agents have been extensively described
in the literature (for a review, see reference 25). For
Grb2, the best results and the most prolonged effect
were obtained with arginine. Some stabilization was
also observed with lysine, taurine, and high concentrations of some salts (ammonium sulfate, sodium
chloride). Surprisingly, widely used agents, such as
glycerol and sugars, had no effect. Arginine is
known to stabilize protein in solutions by increasing
the surface tension and by interacting with negative
charge^.'^ Grb2 has an experimental PI of 5.9 and is
therefore negatively charged a t pH 8.0. The X-ray
structure of the protein7 later proved that the juxtaposition of the two SH3 domains forms a continu-
ous surface a t the bottom of the molecule which presents an alignment of negatively charged residues.
These charges could create strong repulsive interactions between the two SH3. The surface of interaction between them is rather small and these
domains could dissociate and adopt different orientations. So arginine may lower internal electrostatic
repulsions, and thus stabilize a single conformation.
No arginine molecule has been detected in the electronic density of Grb2, at 3 A resolution. Conformational heterogeneity may be rather frequent in
modular proteins, and of course could be very detrimental to their crystallization. In such a case,
screening of stabilizing agents to help freeze internal motions is recommended.
Crystallization
A variety of crystallization conditions and crystallizing agents, including various salts (ammonium
sulfate, sodium phosphate, sodium chloride, potassium thiocyanate, sodium paratoluene-sulfonate),
PEG (400,1,500, and 4,000 kDa), and MPD, was
tested. Experiments were carried out a t 4 and 19°C
in the presence of 100 mM arginine. The first crystals were obtained with 14 mg/ml Grb2, 0.75 M sodium citrate in the pH 8.0 buffer a t 19°C. They grew
overnight. They were small (50 pm in length) and
117
Grb2 CRYSTALLIZATION
twinned. In addition, some protein aggregation
(spherulites) appeared 4 or 5 days after the beginning of the experiments (Fig. 3a).
From a chemical point of view, citrate was the
only carboxylate tested. Its efficiency to crystallize
Grb2 appeared to be linked to the carboxylate functionality, as tartrate and acetate gave the same
small twinned crystals and spherulites. Because citrate and tartrate are strong cation chelating agents
that could later hamper soaking experiments with
heavy atom solutions, all the further crystallization
experiments were done with sodium acetate.
Attempts t o reduce the nucleation rate by optimizing the crystallization conditions yielded larger
crystals. The best crystals (350 pm length) were obtained with 15 mg/ml Grb2,1.7 M sodium acetate, a t
pH 8.0 and 19°C. Nucleation was observed only 2 to
3 days after the beginning of the experiment but the
growth was stopped by the formation of spherulites
2 days later. The overall shape of the crystals was
improved (Fig. 3b). Cutting the twinned crystals in
two and using synchrotron radiation allowed collection of the first X-ray diffraction data.
We succeeded in preventing the formation of spherulites by maintaining a permanent reductive environment for Grb2. This was performed by switching
from vapor diffusion to a dialysis technique. Dialysis
allows the reservoir to be changed daily with renewal
of DTT. Spherulites did not appear, even after several
weeks (Fig. 3c), although crystals were still twinned.
This demonstrated that the origin of the protein aggregation was due to Grb2 oxidation, once DTT had
lost its efficiency. The use of dialysis buttons made it
possible to maintain the necessary reductive potential for several weeks and to pursue the growth experiments over a longer time scale.
In order to improve the shape, size, and quality of
the crystals by decreasing their growth rate,26 we
undertook another series of dialysis experiments
where different percentages of glycerol were added
to the reservoir solutions. Glycerol reduces protein
diffusion in the crystallization solution by increasing viscosity. Indeed, monocrystals appeared after 2
to 4 days with a growth rate of 20 pndday with
6-22% glycerol, sodium acetate, a t pH 8.0 and 19°C.
Nevertheless, most of them soon grew twinned. In a
last optimization step, monocrystals with dimensions around 200 x 200 x 200 pm3 (Fig. 3d) were
obtained with a carefully adjusted acetate concentration and 8 to 14% glycerol. These crystals belong
to the tetragonal space group P4, with a = b = 90.0
8 and c = 97.7 8,and contain two molecules in the
asymmetric unit. They were used for X-ray data collection and enabled the determination of the threedimensional structure of Grb2.7
Glycerol is increasingly being used as an additive
in crystallization trials.27 It has two major positive
effects. First, it prevents the protein from denaturating and aggregating, improving the reproducibil-
ity of the experiments. Second, it improves crystal
quality by decreasing the crystal growth rate and by
reducing twinning. It is sometimes absolutely necessary for crystallization.2s In the case of Grb2, we
found that it had no effect on the stability of the
protein, but that it allowed a better crystallization.
Without glycerol, Grb2 always crystallized in the
form of two embedded twinned crystals. The linking
zone between the two crystalline subunits was more
or less clearly defined depending on the growth
speed. The slower the crystals grew, the better was
their shape.
FINAL REMARKS
SH2 and SH3 domains are conserved protein modules of 100 and 50 amino acids, respectively, found in
a variety of cytoplasmic signaling proteins. The proteins involved in signal transduction often contain
several copies of SH2 and SH3 modules and can thus
be described as modular proteins. Considerable interest lies in their three-dimensional structure analysis. As modular proteins have been proved difficult
to crystallize, and intractable for X-ray crystallographic studies, an alternative “dissect and build
strategy in three steps has been developed for modular protein^.^' First, the structures of isolated
modules are determined, then structural studies on
module pairs provide insight into the way consecutive modules come together. Finally, this information, combined with other low resolution data such
as that obtained by electron microscopy, is used to
rebuild models of intact modular proteins. In the
case of Grb2, starting with the known structure of
the SH2-SH3 domain of Lck,15 the “dissect and
build” method would have predicted relative orientations for the SH2 and SH3 domains different from
the one observed in the 3-D X-ray s t r ~ c t u r e . ~
The crystallization of entire modular proteins is a
difficult challenge. Detailed experimental data and
observations concerning purification, characterization, and crystallization of Grb2 are given here as
they are of more general interest, although the answers for Grb2 (use of DTT, arginine, glycerol) may
not be universal. The strategy described below is
recommended for proteins in general:
(a) high purity is a prerequisite for successful crystallization;
(b) loss of conformational homogeneity can be accurately checked by reverse phase c h r ~ m a t o g r a p h y ; ~ ~
(c) screening of stabilizing agents may allow to select a stable conformation necessary for crystallization;
(d) slowing down crystal growth improves the quality of the crystal;26 and
(e) a careful control of environmental parameters
(redox, pH, T . . .) avoids shifts from their nominal
values.
Points (b) and (c) are specially relevant for rnodu-
Fig. 3. Progressive improvement of the shape of the crystals of Grb2. a: Twinned microcrystals (50 pm in
length) grown from 4 pl hanging drops in the presence of sodium citrate (see text). Most of the protein appears
to be aggregated in the form of spherulites around the crystals. b: Twinned crystal (250 pm in length) grown
from 8 pI, hanging drop in the presence of sodium acetate. c: Twinned crystals grown in a 10 pl dialysis button
in the presence of sodium acetate, with daily addition of fresh D l T in the resewoir. No spherulites appeared,
even after several weeks. d: Mono crystals grown in a 10 pl dialysis button in the presence of sodium acetate
and glycerol.
Grb2 CRYSTALLIZATION
lar proteins, as their difficulty to crystallize is
linked to conformational heterogeneity.
ACKNOWLEDGMENTS
We thank F. Schweighoffer for providing us with
the construction of GST-Grb2. We are greatly indebted to G. Jung for the production of bacteria in
fermentors. We thank B. Monegier for the EMS
analysis. Sebastien Maignan performed the X-ray
characterization of the crystals. This project was
carried out as part of the BioAvenir program supported by Rh6ne-Poulenc with the participation of
the Ministere de 1’EnseignementSuperieur et de la
Recherche and the Ministere de YIndustrie.
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