вход по аккаунту



код для вставкиСкачать
PROTEINS Structure, Function, and Genetics 26353-357 (1996)
Crystallization of the Receptor Binding Domain of
Vascular Endothelial Growth Factor
Hans W.Christinger,' Yves A. Muller,' Lea T. Berleau? Bruce A. Keyt? Brian C. Cunningham,'
Napoleone Ferrara? and Abraham Vos'
Departments of 'Protein Engineering and 2Cardwvascular Research, Genentech, Inc., South Sun Francisco,
California 94080
Vascular endothelial growth
factor (VEGF) is a potent angiogenic factor with
a unique specificity for vascular endothelial
cells. In addition to its role in vasculogenesis
and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four
different constructs of a short variant of VEGF
sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified,
and crystallized in five different space groups.
In order to facilitate the production of heavy
atom derivatives, single cysteine mutants were
designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space
group P2,,with cell parameters a = 55.6 A, b =
60.4 A, c = 77.7 A, p = 90.0", and four monomers
in the asymmetric unit. Native and derivative
data were collected for two of the cysteine mutants as well as for wild-type VEGF.
1996 Wiley-Liss, Inc.
Key words: VEGF, angiogenesis, tumor vascularization, inclusion bodies, cysteine mutants, X-ray crystallography, crystals
Angiogenesis (the development of new blood vessels) is required for many physiological processes
such as embryogenesis, wound healing, and corpus
luteum formation. Vascular endothelial growth factor (VEGF), also known as vascular permeability
factor (VPF), is a highly endothelial cell-specific angiogenic factor and has been implicated in the neovascularization of
Although several
other potential mediators of tumor vascularization,
including basic and acidic fibroblast growth factors,
tumor necrosis factor-a, and transforming growth
factors-a and -p, have been identified in situ, these
factors lack specificity for endothelial cells. The ap0 1996 WILEY-LISS, INC.
parent involvement of VEGF in diabetic retinopathy
and other retinal neovascular disorder^,^ as well as
the inhibition of tumor growth in vivo by antibodies
directed against VEGF,' make it a prime candidate
as the mediator of normal and pathological angiogenesis. Therefore, VEGF antagonists will potentially be useful therapeutic agents to regulate excessive vascularization.
VEGF is a homodimeric glycoprotein which naturally occurs in four different isoforms, each monomer having 121, 165, 189, or 206 amino acids
(VEGF-121, VEGF-165, VEGF-189, and VEGF206). The different isoforms result from alternative
C-terminal splicing and whereas VEGF-121 lacks
affinity for heparin, all other isoforms are highly
basic proteins tightly bound to extracellular heparin-containing prote~glycans.~
The absence of the
heparin binding domain does not reduce the binding
affinity of VEGF to its receptors KDR and FLT, but
does decrease the activity of VEGF in a cell-based
assay, suggesting that the role of the heparin binding domain is to sequester soluble VEGF to the cell
surface.* A shorter isoform of VEGF retaining full
receptor binding affinity can be obtained in vivo
upon plasmin cleavage after residue 110 (VEGF110).8
Sequence homology suggests that the receptorbinding domain of VEGF (residues 1 to 110) is a
member of the cystine-knot fa mil^.^*'^ This family is
characterized by three disulfide bridges forming a
knot-motif. Other members of this family with
known three-dimensional structure are nerve
growth factor," transforming growth f a ~ t o r - p , ' ~ , ~ ~
platelet-derived growth factor (PDGF),14and chorionic g~nadotropin.'~,~'
These molecules all form
dimers, but even though the monomer units share a
conserved fold, the dimer interfaces are completely
Received March 29,1996; revision accepted April 5, 1996.
Address reprint requests to Abraham M. de Vos, Department
of Protein Engineering, Genentech, Inc., 460 Point San Bruno
Boulevard, South San Francisco, CA 94080.
different? With the exception of the cysteine residues, VEGF has very little sequence homology to
the other members of the family; the connectivity
of intermolecular disulfide bridges suggests that
VEGF is most closely related to PDGF.'
Due to the central role of VEGF in angiogenesis
and in order to better understand its biological activity on a molecular level, we decided to determine
the crystal structure of the receptor binding domain
of VEGF. Here we report the purification, refolding,
and crystallization of four different VEGF variants
(VEGF-8~109,VEGF-110, VEGF-121, VEGF-1651,
undertaken because of our initial failure to obtain
suitable crystals for some of the variants. In addition, when production of heavy atom derivatives using conventional soaking methods proved unsuccessful, we designed mutants each with a single
residue replaced by cysteine. The success of these
approaches demonstrates the power of the application of molecular biology methods in support of
structural studies.
Two isoforms of VEGF (VEGF-121 and VEGF165), an artificial construct consisting of residues 8
to 109 (VEGF-8:109), and two single cysteine mutants (P53C and Q98C) of the latter were expressed
as insoluble proteins in E. coli in amounts of 60 to 80
mg per 100 g of cell paste. Inclusion bodies were
isolated by passing homogenized cells in 20 mM
TRIS pH 7.5, 5 mM EDTA through a French pressure cell, and centrifuging the homogenate for 15
minutes at 4,OOOg. The inclusion bodies were then
resuspended and centrifugation repeated. The pellet
consisting predominately of VEGF was dissolved in
7.5 M urea, 20 mM TRIS pH 7.5, and stirred for 1
hour in 20 mM dithiothreitol (DTT) in order to
achieve complete reduction of incorrectly formed disulfide bridges. VEGF-121, VEGF-165, and VEGF8:109 were refolded by diluting the above solution to
a final concentration of about 0.75 mg/ml and then
dialyzing for 24 hours against 20 mM TRIS-HCl
(pH 8.4), 0.4 M sodium chloride, 1 mM cysteine.
Identical results were obtained with 7 pM CuC1, in
the refolding buffer. The single cysteine mutants
were refolded in the absence of free cysteine and
The refolded proteins VEGF-121 and VEGF-8:109
were purified to homogeneity in consecutive steps of
ion exchange chromatography (Q-Sepharose; Pharmacia, Uppsala, Sweden), hydrophobic interaction
chromatography (Alkyl-Sepharose; Pharmacia) and
size exclusion chromatography (S-100; Pharmacia).
In order to protect the free cysteines in the cysteine
point mutations of the VEGF-8:109 variant, the first
two purification steps were performed in the presence of 2 mM D'I"I'. The highly basic protein VEGF165 was purified by ion exchange chromatography
(S-Sepharose, Pharmacia) followed by a copper chelating column and a second ion exchange chromatography column (SB-Toyo Pearl, Toso Haas, Philadelphia, PA). Portions of the protein were then
subjected to plasmin cleavage in order to generate
VEGF-110.' VEGF-110 was purified by negative absorption to a heparin affinity column followed by ion
exchange chromatography (Q-Sepharose; Pharmacia). The purity and homogeneity of all proteins
were monitored by SDS-PAGE and mass spectrometry. In addition, receptor-binding activity to the extracellular domain of the KDR receptor was tested
with IgG-KDR fusion proteins in a radio receptor
Initial screening for crystallization conditions was
done according to the sparse matrix method17 using
commercially available buffers (Hampton Research,
Riverside, CA), suspending droplets obtained after
mixing 2 p1 of protein solution [lo-15 mg/ml protein
in crystallization buffer (20 mM TRIS-HCl, pH 7.5,
0.4 M sodium chloride)] with 2 pl of reservoir solution over 900 pl of reservoir solution in cell culture
plates from Linbro (Flow Laboratories, McLean,
VA). After optimization of the initial crystallization
conditions, large single crystals were grown in sitting drops consisting of 40 pL protein mixed with 40
pL reservoir solution in nine-well sitting-drop trays
from Corning (Corning, NY).
All crystallographic data were recorded in-house
using graphite-monochromated CuKa radiation
generated from a Rigaku RU200 rotating anode
(Rigaku-USA, Danvers, MA) operating at 45V,
12OmA, and a MAR Research imaging plate (MAR
Research, Hamburg, Germany) with a 300 mm diameter. Usually data were recorded in 1" rotation
steps with an exposure time of 15 minutes and indexed using the automatic indexing routines of programs XDS1' and DENZO."
Design of Cysteine Mutants
When conventional attempts at producing derivatives were unsuccessful, single cysteine mutations
were introduced into VEGF. The design of the mutations was achieved by displaying the sequence of
VEGF onto the backbone of the existing PDGF
structure14 and looking for partially exposed side
chains in well-ordered portions of the model. In order to prevent misfolding of the cystine-knot disulfide bridges, sites chosen were distant in space from
existing cysteine residues. No restrictions were imposed on the amino acid type a t the residues considered for mutation. We selected a final set of four
potential mutation sites (Pro53, Thr71, Leu97,
Gln98) distributed evenly over the molecule (Fig. 1);
two of the mutants were crystallized, but in practice,
a single mutant (P53C) was sufficient to determine
the structure.
Fig. 1. Backbone (in gray) and disulfide bonds (in green) of the
PDGF dimer, showing VEGF side chains for residues selected for
substitution with cysteine in yellow. Residues on one monomer
are labeled using VEGF numbering. The Cys53 and Cys98 point
mutants were expressed, refolded and purified, and crystallized
isomorphously with wild-type VEGF.
Purification and Refolding
Mass spectrometry confirmed the correct mass
and homogeneity of the purified variants. All constructs showed similar receptor binding activity
(Bing Li, personal communication). The final yield
of purified wild-type VEGF was about 30 to 40 mg
per 100 g of cell paste (approximately 50%); some of
the losses represented VEGF running anomalously
on the hydrophobic interaction chromatography column, and we assume this was misfolded protein. Initial purification and refolding attempts for the single cysteine mutants using the refolding protocol
developed for the wild-type variants failed, as in all
cases the determined mass differed from the expected mass. The mass difference could be accounted
for by cysteine molecules from the refolding buffer
bound to the free cysteines of the mutants. In other
attempts, observed mass differences suggested partial oxidation by molecular oxygen of the free sulfhydryl groups of the mutants. Only after using DTT
in the refolding buffer and maintaining reducing
conditions during the first two purification steps,
could the correct masses be observed for the single
cysteine substitutions. After re-optimization of the
refolding and purification protocols, the yield of the
mutants increased from 10 to 20 mg per 100 g of
paste to the levels observed for wild-type VEGF.
Dithionitrobenzoate assays showed two free sulfhydry1 groups per VEGF dimer.
Crystallizationof VEGF-165, VEGF-121,
and VEGF-110
Crystallization attempts with the VEGF-165 variant resulted in the formation of poorly diffracting
crystals which could not be improved. X-ray quality
crystals were first obtained using VEGF-110 generated by plasmin cleavage of VEGF-165; the same
space groups were later observed for VEGF-121.
Hexagonal rods grew aRer mixing 40 p1 of protein in
crystallization buffer with 40 p1 of reservoir solution
(0.2 M ammonium sulfate, 2.0 M sodium formate in
0.1 M sodium acetate buffer pH 4.6) and equilibrating against reservoir solution (Fig. 2a). The space
group was determined as P61(5j (a=96.9 A, c = 127.2
A), and considerations of solvent content suggested
two VEGF dimers in the asymmetric unit (VM= 3.4
b3/Da). However, the crystals diffracted only to
about 4 A and were sensitive to X-ray exposure. In
one case, a spontaneous transition from P6, 5 )
(a=96.9 A, c=127.2 A) to P6,(,,22 (a=96.9
c = 63.6 A1 was observed upon radiation exposure.
A second crystal form consisting of orthorhombic
Fig. 2. Five different crystal forms obtained with different constructs of the receptor binding domain of VEGF. a: Hexagonal
rods (space group p6,(,,; b: orthorhombic blocks (R,2,2,) obtained with VEGF-110 and VEGF-121; c: monoclinic
d: tri-
clinic (Pl) crystals obtained with VEGF-8:109; e: monoclinic crystals (F2,) of VEGF-8:109 used to determine the structure by multiple isomorphous replacement.
blocks originally grew using a reservoir solution
consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate, 30% polyethylene glycol (PEG) 3350,
pH 4.6 (Fig. 2b). These crystals were determined to
be of space group P2,2,2, (a=48.2 b, b=61.5 A,
c= 186.2 A), diffracted to 3.3 b resolution and contained two dimers per asymmetric unit (V, = 2.7 b31
Da). After failing to determine the structure by molecular replacement using the PDGF structure as a
search model, an extensive search for heavy atom
derivatives was undertaken. However, soaking with
heavy atom solutions resulted in large changes in
the cell axes (>5%)and no stabilizing mother liquid
could be found.
40% 2-methyl-2,4-pentanediol
(MPD) in 0.1M MES
buffer pH 6.0 (Fig. 2c). The crystals grew within 2
weeks and diffracted to better than 3.3 A resolution.
Assuming a V
, of 2.0 b3/Da, the crystals contained
four dimers in the asymmetric unit. Crystals in
space group P1 (a=45.9 A, b=68.7 b, c=85.2 A,
a = 104.9", f3 =90.0", y = 101.7') were obtained from
droplets equilibrating against a reservoir of 14%
PEG3350, 10% isopropanol, 0.2 M ammonium acetate pH 5.6 (Fig. 2d). Assuming a V, of 2.7 A3/Da,
the crystals contained four dimers in the asymmetric unit. The crystals diffracted to better than 3.0 b
A third crystal form was obtained by adding calcium chloride to a condition that had previously resulted in precipitate. Crystals in space group P2,
(a= 55.6 A, b = 60.4 A, c = 77.7 A, f3 = 90') grew from
30% PEG3350, 20% isopropanol, 0.2M ammonium
acetate, 0.2M calcium chloride (pH 5.6), reaching a
size of 0.6 x 0.3 x 0.3 mm3 within 2 days (Fig. 2e).
Decreasing the PEG concentration slowed crystal
growth and improved crystal morphology, and conditions were further improved by adding sodium
chloride. The final conditions were 14% PEG3350,
20% isopropanol, 0.2M ammonium acetate, 0.2M
calcium chloride, 0.4M sodium chloride (pH 5.6).
Stabilizing artificial mother liquor was found by reducing the isopropanol concentration to 5%, adding
5% MPD, and increasing the PEG concentration to
Crystallization of VEGF-8109
Preliminary experiments using nuclear magnetic
resonance spectroscopy to determine the three-dimensional structures of the receptor-binding domain of VEGF suggested that the first seven residues, as well as the last residue of VEGF-110, are
disordered in solution (W.J. Fairbrother, personal
communication). Therefore, a shortened variant,
VEGF-8:109, was constructed and used in crystallization screenings, resulting in a total of three different crystal forms suitable for crystallography.
Crystals in space group C2 (a=235.5 A, b=44.4
b, c = 75.2 A, p = 101.6") were grown from sitting
drops equilibrating against reservoir containing
30%. Reducing the isopropanol concentration from
20 to 5%greatly simplified the handling of the crystals as the evaporation of isopropanol caused severe
convection in the drops as a result of concentration
and temperature gradients.
The crystals contained two dimers in the asymmetric unit (V, = 2.7 A3iDa) and diffracted anisotropically to better than 2.5 A in the (a*,c*) plane
but only to 2.7 A in the b* direction. The search for
heavy atom derivatives was again hindered by nonisomorphism reflected in large changes in the b cell
axis upon soaking the crystals with heavy atom solutions. However, we could readily obtain isomorphous crystals of the single cysteine mutants in this
space group, enabling us to produce a heavy atom
derivative by soaking the mutant crystal with lead
acetate and using an unsoaked mutant crystal for a
native dataset. “he solvent-flattened single isomorphous replacement density clearly allows for the determination of the relationships between monomers
in the asymmetric unit, and structure determination making extensive use of non-crystallographic
symmetry is in progress.
We thank Jim Wells and Tony Kossiakoff for support and comments on the manuscript; Wayne Fairbrother for suggesting the 8-109 construct; Dot
Reilly and Sylvia Wong for fermentation runs;
Henry Heinsohn, Rex Hayes, Marge Winkler, Mark
Ultsch, and Mike Randal for suggestions on refolding and purification; Beth Gillece-Castro and Jim
Bourell for mass spectrometry data; Angie Namenuk and Alan Padua for N-terminal sequencing;
and Bing Li for binding assays.
1. Ferrara, N. The role of vascular endothelial growth factor
in phatological angiogenesis. Breast Cancer Res. Treat.
36:127-137, 1995.
2. Folkman, J . Angiogenesis in cancer, vascular, rheumatoid
and other disease. Nature Med. 1:27-31, 1995.
3. Dvorak, H.F., Brown, L.F., Detmar, M., Dvorak, A.M. Vascular permeability factorhascular endothelial growth factor, microvascular hyperpermeability and angiogenesis.
Am. J. Pathol. 146:1029-1039, 1995.
4. Neufeld, G., Tessler, S., Gitay-Goren, H., Cohen, T., Levi,
B.Z. Vascular endothelial growth factor and its receptors.
Prog. Growth Factor Res. 5:89-97, 1994.
5. Aiello, L.P., Avery, R.L., Arrigg, P.G., Keyt, B.A., Jampel,
H.D., Shah, S.T., Pasquale, L.R., Thieme, H., Iwamoto,
M.A., Park, J.E., Nguyen, H., Aiello, L.M., Ferrara, N.,
King, G.L. Vascular endothelial growth in occular fluid of
patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331:1480-1487, 1994.
6. Kim, K.J., Li, B., Winer, J., Armanini, M., Gillet, N., Phillips, H.S., Ferrara, N. Inhibition of vascular endothelial
growth factor-induced angiogenesis suppresses tumour
growth in vivo. Nature 362:841-844, 1993.
7. Park, J.E., Keller, G.A., Ferrara, N. The vascular endothelial growth factor (VEGF) isoforms: Differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol. Biol.
Cell. 4:1317-1326, 1993.
8. Keyt, B.A., Berleau, L.T., Nguyen, H.V., Chen, Heinsohn,
H., Vandlen, R., Ferrara, N. The carboxyl-terminal domain of vascular endothelial growth factor is critical for its
mitogenic potency. J. Biol. Chem. 271:7788-7795, 1996.
9. Sun, P.D., Davies, D.R. The cystine-knot growth-factor superfamily. Annu. Rev. Biophys. Biomol. Struct. 24269291,1995.
10. Isaacs, N.W. Cystine knots. Curr. Opin. Struct. Biol.
5:391-395, 1995.
11. McDonald, N.Q., Lapatto, R., Murray-Rust, J., Gunning,
J., Wlodawer, A., Blundell, T.L. New protein fold revealed
by a 2.3 A resolution crystal structure of nerve growth
factor. Nature 354:411-414, 1991.
12. Schlunegger, M.P., Grutter, M.G. Refined crystal structure of human transforming growth factor-p2 at 1.95 A
resolution. J. Mol. Biol. 231:445-458, 1993.
13. Daopin, S., Li, M., Davies, D.R. Crystal structure of
TGF-p2 refined at 1.8 A resolution. Proteins 17:176-192,
14. Oefner, C., D’Arcy, A., Winkler, F.K., Eggimann, B., Hosang, M. Crystal structure of human platelet-derived
growth factor BB. EMBO J. 11:3921-3926, 1992.
15. Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader,
J.W., Canfield, R.E., Machin, K.J. Morgan, F.J., Isaacs,
N.W. Crystal structure of human chorionic gonadotropin.
Nature 369:455-461, 1994.
16. Wu,H., Lustbader, J.W., Liu, Y., Canfield, R.E., Hendrickson, W.A. Structure of human chorionic gonadotropin at
2.6 A resolution from MAD analysis of the selenomethionyl protein. Structure 2:545-558, 1994.
17. Jancarik, J., Kim, S.H. Sparse matrix sampling: A screening method for crystallization of proteins. J . Appl. Crystallogr. 24:409-411, 1991.
18. Kabsch, W. Evaluation of single crystal X-ray diffraction
data from a position sensitive detector. J. Appl. Crystallogr. 21:916-924, 1988.
19. Otwinowski, 2. Oscillation data reduction program. In:
Proceedings of the CCP4 Study Weekend “Data Collection
and Processing.” Sawyer, L., Isaacs, N., Bailey, S. (eds.).
UK: SERC Daresbury Laboratory, Warrington, 1993:5662.
Без категории
Размер файла
530 Кб
Пожаловаться на содержимое документа