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Research Article

Received: 10 December 2009, Revised: 23 February 2010, Accepted: 24 February 2010, Published online in Wiley Online Library: 4 May 2010

(wileyonlinelibrary.com) DOI:10.1002/jmr.1039

The testosterone binding mechanism of an


antibody derived from a naı̈ve human scFv
library
Merja H. Niemia, Kristiina Takkinenb, Lotta K. Amundsenb,
Hans Söderlundb, Juha Rouvinena * and Matti Höyhtyäc
A testosterone binding scFv antibody was isolated from a naı̈ve human library with a modest size of 108 clones. The
crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å
resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with
framework residues only CDR-L3 and CDR-H3 loops interact with testosterone and the heavy chain forms the majority
of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic
amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher
affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few
light chain contacts. This is the first three-dimensional structure of a human steroid hormone binding antibody that
has been isolated from a naı̈ve human repertoire. Copyright ß 2010 John Wiley & Sons, Ltd.

Keywords: antibody library; crystal structure; fab fragment; hapten; steroid hormone; twinning

INTRODUCTION We have earlier generated a highly selective, high affinity


(Kd 3  1010 M) recombinant anti-testosterone Fab fragment,
Antibody phage display libraries combined with powerful in vitro 77 Fab, by stepwise optimization of the complementarity
selection techniques are currently widely used for generating determining regions (CDRs) of a monoclonal antibody 3-C4F5
antibodies against desired therapeutic or diagnostic targets (Hemminki et al., 1998a,b). The crystal structures of the mutant 77
(Hoogenboom, 2005). Steroid hormones, which are important and wt (3-C4F5) Fab fragments with and without testosterone
clinical markers of hormonal disorders, are one class of analytes revealed that the significantly improved affinity and specificity of
for which antibodies have been developed by the recombinant the 77 Fab fragment is based on the more comprehensive
techniques during recent years, due to the fact that the packing of the testosterone with the protein, which is the result of
development of monoclonal antibodies with high enough small structural changes within the variable domains (Valjakka
specificity and affinity has not been successful through the et al., 2002a,b).
use of hybridoma technology. Steroids are small, rigid, In this study, we isolated a testosterone binding scFv antibody
hydrophobic molecules with only a few functional groups 5F2 from a naı̈ve human scFv library. The three dimensional
capable of specific interactions with antibodies. The number of structure of the 5F2 antibody in the Fab fragment format in
different closely related steroids in human serum is high, their in complex with testosterone was solved. 5F2 clone has the modest
vivo concentrations are low (down to picomolar level), and their testosterone binding affinity (Kd  107 M). However, the 5F2 Fab
relative concentrations can vary greatly, even between normal derived from an unimmunized source and the high affinity 77 Fab
healthy individuals. evolved from a monoclonal antibody show similar binding
Steroid hormone binding antibodies have been isolated from features.
human naı̈ve scFv libraries of different sizes (Vaughan et al., 1996;
Dörsam et al., 1997; Krebs et al., 2001). The high affinity and
specificity requirements needed for reliable measurement of
steroid hormone levels, however, have not been reached with the * Correspondence to: J. Rouvinen, Department of Chemistry, University of
Eastern Finland, P.O. Box 111, FIN-80101 Joensuu, Finland.
antibodies isolated from the libraries. Using a large naı̈ve scFv
E-mail: juha.rouvinen@uef.fi
library (1.38  1010 clones) Pope et al. (1996) were able to isolate an
estradiol binding scFv antibody with a nanomolar range affinity, a M. H. Niemi, J. Rouvinen
which is comparable to affinities of monoclonal antibodies. The Department of Chemistry, University of Eastern Finland, P.O. Box 111,
other strategy for isolating steroid binders has been the use of FIN-80101 Joensuu, Finland
designed focused antibody libraries against haptens (Persson et al., b K. Takkinen, L. K. Amundsen, H. Söderlund
2006), but in this case testosterone binding scFv clones with VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT,
modest affinities (Kd  107 M) were isolated. By exploiting in vitro Finland
evolution, the affinity of one testosterone binding clone was c M. Höyhtyä
further improved 200-fold (Persson et al., 2008). MedixBiochemica, Asematie 13, FIN-027700, Kauniainen, Finland
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J. Mol. Recognit. 2011; 24: 209–219 Copyright ß 2010 John Wiley & Sons, Ltd.
M. H. NIEMI ET AL.

MATERIALS AND METHODS 15 min 48C) and the culture supernatant concentrated to a final
volume of 1 L using a Pellicon concentrator (Millipore) and a
Isolation of the 5F2 anti-testosterone clone cellulose filter with a 10 kDa cut-off (Millipore). During the
concentration, the buffer was exchanged to PBS (pH 7.4). The Fab
The VTT naı̈ve human scFv phage display libraries VHVk and VHVl fragment was purified using a Cu2þ-Chelating Sepharose
(both containing approximately 108 clones) have been cloned Streamline column (Amersham-Pharmacia) and a linear gradient
from a pool of human lymphocytes of 50 healthy donors elution (20–500 mM imidazole in PBS, pH 7.4). Fractions
(Reinman et al., 2003; Pulli et al., 2005; Weber et al., 2005; Turunen containing the Fab fragment were pooled and the buffer was
et al., 2009). scFv libraries were enriched against a testoster- exchanged to PBS.
one-3-CMO-BSA conjugate (TES–BSA, Sigma) in two panning The affinity of 5F2 Fab was determined by ELISA by coating
rounds. Briefly, libraries were electroporated into XL-1 blue microtiter plate wells with 0.3 mg of TES–BSA in a 0.1 M bicarbonate
Escherichia coli cells, and phage particles were rescued by buffer (pH 9.6) for 16 h at 48C. The wells were then washed three
infection of the bacteria with the helper phage VCSM13 times with PBS and blocked with 200 ml of PBS–0.5% BSA for 1 h at
(Stratagene). Libraries were incubated for 18 h at þ48C in BSA RT and washed. The samples containing 5F2 Fab with soluble
coated wells (to deplete the BSA binders) and transferred into testosterone were prepared by mixing 900 ng Fab 5F2 in 450 ml of
(TES–BSA, 1 mg/100 ml/well) coated wells. After 1 h incubation at PBS-0.5% BSA and 50 ml of increasing concentrations of testoster-
RT, the wells were washed 20 times with PBS and the bound one (0, 106, 105, 104, 103, 102, 101, 1, 10, 100 and 10 000 mM)
phages were eluted with 100 mM glycine–HCl buffer pH 2.2 and in PBS in test tubes. An aliquot of 100 ml of each sample was
after neutralization used to reinfect E. coli. pipetted into wells, four parallel measurements were made. The
samples were incubated at room temperature for 1 h. After the
ELISA screening of E. coli colonies producing scFv-fragments washing step (three times with PBS), the detection was carried out
After the second selection round, single colonies were picked by with a goat anti-human F(ab)2 alkaline phosphatase conjugate
the QPix colony picker (Genetix) into 96-well plates containing (Rockland) diluted by 1:2000 in PBS–0.5% BSA. After a 1 h incubation
200 ml/well of SB-media with 100 mg/ml of ampicillin, 10 mg/ml of at RT, the wells were washed and 100 ml of 2 mg/mL of
tetracycline and 1% glucose. The colonies were grown o/n at p-nitrophenylphosphate (Sigma) in diethanolamine–MgCl2 buffer
þ308C with 700 rpm and 85% humidity in a shaker (InforsMulti- (Reagena) was added to the wells. The absorbance was measured at
tron). 150 ml of the culture suspension was transferred into new 405 nm after 30 min (Varioscan, Thermo Electron Corporation).
plates. The o/n cultivation plates containing the rest of the
inoculm cultivation (50 ml) were stored as the master plates at Crystallization and X-ray data collection
48C. Culture suspensions transferred to the new plates were
centrifuged for 10 min at 2000 g (Eppendorf Centrifuge 5810R) The anti-testosterone 5F2 Fab fragment was co-crystallized
and the culture supernatant was removed. The cells were with testosterone by using the hanging-drop vapour-diffusion
resuspended into fresh SB-media (200 ml) supplemented with method. Favourable crystallization conditions were found using
ampicillin (100 mg/ml), tetracycline (10 mg/ml) and 1 mM iso- the modified PEG3350 series for antibodies (Valjakka et al., 2000).
propyl b-D-thiogalactopyranoside and the plates were grown for 5F2 Fab crystals suitable for X-ray analysis were obtained by
20 h, as before. The binding activities of the soluble scFv–myc–pIII combining the streak-seeding method with nanospheres (Kallio
fusion proteins released into the culture supernatant were et al., 2009). Briefly, the crystallization droplets contained 2 ml of
analysed by ELISA. As a control for the screening assay, the protein solution (8.3 mg/ml in 20 mM HEPES buffer, pH 7.0), 0.5 ml
anti-testosterone 77 Fab fragment was inoculated into four wells of testosterone solution (5 mM in 50% ethanol), 1 ml of nano-
per plate and used as a control for the testosterone binding. ELISA sphere solution (50 nm in diameter, 1:1 dilution to water) and 2 ml
was performed using TES–BSA coated wells and mouse anti-myc of precipitant solution (12% v/w PEG3350, 0.1 M sodium citrate
antibody (clone 9E10, ATCC # CRL-1729) and goat anti-mouse at pH 4.7). The protein droplets were allowed to pre-equilibrate
IgG-AP conjugated antibody (Bio-Rad) for detection (detecting for 24 h and were then seeded by transferring crystal nuclei from
also the expressed 77 Fab fragment). previously grown 5F2 Fab crystal clusters. Single crystals reached
The scFv coding regions of the positive clones were sequenced the approximate dimensions of 0.2  0.1  0.1 mm3.
by an ABI 3100 Genetic Analyser using a BigDye 3.0 Sequence For data collection, the crystals were transferred to a
Termination Kit (Applied Biosystems). cryoprotective solution (12% PEG3350, 20% glycerol, 0.1 M
sodium citrate). After cryoprotection, crystals were frozen by
plunging them into liquid nitrogen. The X-ray data were collected
Production, purification and characterization of the
from a single crystal that diffracted to a resolution of 1.5 Å on the
anti-testosterone 5F2 Fab fragment
beamline ID-29 at the ESRF (Grenoble, France). The crystal was
For further characterization, the 5F2 scFv was converted to a Fab exposed for 2 s with a 1.08 oscillation per frame. Reflections were
fragment by cloning the genes encoding human IgG1 CH1- and recorded with an ADSC Q315R detector. The diffraction data were
Ck-constant regions in frame with VH- and VL-regions, respect- processed with XDS and scaled with XSCALE (Kabsch, 1993).
ively. The genes encoding the 5F2 Fab fragment were cloned into
the E. coli expression vector (pKKtac) containing a hexahistidine
Structure solution from twinned 5F2 Fab crystal
tag at the C-terminus of the heavy chain constant region for
immobilized metal ion affinity chromatography (IMAC)- The data were initially processed in the monoclinic space group
purification. High cell density cultivation of the 5F2 Fab fragment C2 with the unit cell parameters a ¼ 101.7, b ¼ 87.4, c ¼ 66.7 Å
was carried out in a 4.5-L fermenter using E. coli RV308 strain and b ¼ 117.88. The diffraction intensity statistics analysis
(ATCC 31608), as described (Nevanen et al., 2001). After the performed with the program TRUNCATE from the CCP4 package
cultivation, the cells were harvested by centrifugation (3963  g, (Collaborative Computational Project, Number 4, 1994) did not
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THE TESTOSTERONE BINDING MECHANISM

indicate twinning. The cumulative intensity distribution for


Table 1. Statistics of data collection and refinement
acentric reflections followed the theoretical curve for untwinned
data. In addition, the second intensity moment (I2)/(I)2 for acentric
reflections was 2.19, as expected values are 2.0 for normal data Data collection
and 1.5 for perfectly twinned data. Space group P1
The structure was solved in space group C2 with the molecular Wavelength (Å) 1.00911
replacement method. The VLA-1 integrin 1 domain binding Fab Resolution range (Å) 50–1.5 (1.6–1.5)
fragment of humanized AQC2 antibody (Karpusas et al., 2003, PDB Unit cell parameters
accession code 1MHP) was used as the search model. The sequence a, b, c (Å) 66.8, 67.1, 67.1
identities were 75% for the light chain and 91% for the heavy chain. a, b, g (8) 81.3, 69.3, 69.3
The molecular replacement was carried out with Molrep (Vagin and Unique reflections 151891 (26151)
Teplyakov, 1997). The constant and variable domains of model Completeness (%) 93.1 (91.0)
protein were used separately to determine rotation and translation Average I/s 14.0 (2.5)
functions for 5F2 Fab fragment. The molecular replacement gave a Robs (%) 3.3 (32.1)
clear solution for one 5F2 Fab molecule in an asymmetric unit. The Rmeas (%) 4.7 (45.3)
Matthews coefficient (Matthews, 1968) calculated for one 5F2 Fab Refinement
molecule in the monoclinic C2 space group was 2.62 Å3/Da, R/Rfree (%) 17.9/20.8
corresponding to a solvent content of 53%. Twin fraction 0.5
After several refinement and model building cycles, the No. of atoms/B factors 7532/20.1
difficulties with the data emerged. The difference electron Protein 6571/19.7
density map for the heavy chain regions of Pro15H-Ser36H and Hapten 42/17.7
Ser54H-Arg95H showed two favourable positions for the Water 919/23.3
polypeptide chain with a distance of 1.4 Å. In the monoclinic Rms deviations
cell, these regions were located near the crystallographic twofold Bond lengths (Å) 0.013
axis. The R and Rfree factors (22.6 and 26.1%, respectively) of the Bond angles (8) 1.658
final model in the C2 cell were also relatively high for the 1.5 Å data. Numbers in parentheses refer to the highest resolution shell.
The data were re-indexed in the space group P1. Coincidentally,
two edges and two angles of the P1 unit cell were approximately
equal, thus allowing pseudo-merohedral twinning. Similar pseu- antibodies were isolated by screening 440 individual E. coli clones
do-merohedral twinning in the P1 space group has been reported (data not shown). The 5F2 scFv clone, showing a relatively good
to appear in a RNA fragment crystal (Mueller et al., 1995). Using the binding affinity, in the primary binding profile characterizations,
previously refined 5F2 Fab structure as a search model, the was selected for further characterization and was cloned into the
positions of two 5F2 Fab molecules in the P1 cell were determined Fab fragment format. The 5F2 Fab fragment was produced in 4.5 L
by molecular replacement. The twin operator h, l, k was fermenter and purified with IMAC. The testosterone binding affinity
defined by phenix.xtriage and the twin refinement was carried out of the 5F2 Fab fragment is 900 nM as determined by a competitive
by phenix.refine (Adams et al., 2002). Refinement in space group P1 ELISA (Figure 1B). Binding specificity of the 5F2 Fab was also
with the introduced twin law and anisotropic correction of maps studied by using 5a-dihydrotestosterone, dehydroepiandroster-
lowered both the crystallographic R-factors and improved the one sulphate and androstenedione as the soluble competitors. No
quality of the electron density maps. When the twinning was taken inhibition was obtained with these ligands in the concentration
into account, R and Rfree factors dropped to values of 18.0 and range of (0–100 mM). This indicates a very low or no binding affinity
20.8%, respectively. The twin fraction was 0.5, corresponding to a of 5F2 Fab towards these steroids (data not shown).
perfectly twinned crystal (Table 1).
The final protein model was validated with an ADIT validation
Overall structure of 5F2 Fab
server (Westbrook et al., 2003) on RCSB web sites. In the
Ramachandran plot, 99.2% of the amino acid residues lied in The three-dimensional structure of the 5F2 Fab fragment in
most favoured or additional allowed regions and 0.3% of the complex with testosterone was determined at a resolution of 1.5 Å.
residues were in disallowed regions. Buried surface areas Protein was crystallized in the space group P1 with two 5F2 Fab
were calculated by the AreaIMol programme (CCP4) with a molecules in an asymmetric unit. The variable and the constant
probe radius of 1.4 Å and the antibody-hapten contacts domains of the 5F2 Fab fragment followed the typical immu-
were evaluated by NCONT (CCP4). The cut-off value for the noglobulin fold (Figure 2). Owing to the weak electron density, two
determination of van der Waals interactions was 4.0 Å. The N-terminal residues of the light chain and two C-terminal residues
coordinates and the structure factors have been deposited in of the heavy chain were excluded from the final protein model. The
the RSCB Protein Data Bank with the accession code of 3KDM. electron density maps were also unclear for the loop in the heavy
chain region of Ser1010H-Gly1016H (IMGT numbered residues,
Lefranc et al., 2005). This loop is frequently disordered in antibody
structures. Based on the electron density, the cyclic pyroglutamic
RESULTS acid was placed at the N-terminus of the heavy chain.
Isolation of the 5F2 anti-testosterone clone
Complementarity determining regions
ELISA based screening was used for the isolation of scFv clones
from the second round selection pools of the VTT naı̈ve human The positions and the lengths of the CDRs were defined according
scFv (Figure 1A). Altogether, 12 testosterone binding scFv to IMGT unique numbering scheme for immunoglobulin variable
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J. Mol. Recognit. 2011; 24: 209–219 Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr
M. H. NIEMI ET AL.

Figure 1. Screening individual scFv clones and analysis of the binding affinity of the 5F2 Fab by ELISA. (A) The culture supernatant samples of 92
individual clones producing soluble scFv–myc–pIII fusion proteins and four anti-testosterone 77 Fab producing colonies (marked with a black circle) were
analysed on TES–BSA coated wells. Five scFv clones (marked with an asterisk) showing higher binding response than 77 Fab were selected for further
characterization. (B) The percentage of the total binding represents the value obtained by dividing the mean binding signal, an average of four parallel
measurements, at each testosterone concentration by the binding response without the competing testosterone. The IC50value of 5F2 Fab, equal to the
testosterone concentration at which the binding response is 50% of the maximum, is 900 nM.

domains (Lefranc et al., 2003). The light chain of 5F2 Fab belongs to side-chain of Trp118H and the main-chain oxygen of Met115H
the l class. All three CDR loops of the light chain and the CDR-1 and stabilize the bulged conformation in the torso region (Morea et al.,
CDR-2 loops of the heavy chain could be classified into the 1998). The tip of the CDR-H3 loop contains two hydrophobic
common canonical structures of immunoglobulins (Al-Lazikani residues, Leu110H and Trp111H, which form a hydrophobic patch
et al., 1997). CDR-L1 loop (9 residues) represents canonical class 2 on the antibody surface.
with a slightly distorted a-helix. CDR-L2 loop consists of three
residues and belongs to canonical class 1. CDR-L3 contains 11
Testosterone binding site
residues and resembles the structures of canonical class 2, though
Pro115L breaks the typical hydrogen bond pattern. The heavy Testosterone is bound in a deep pocket, which is located between
chain CDR-H1 (8 residues) has canonical structure 1. Arg80H from the variable domains of the light chain and the heavy chain of an
b-strand 4 is important for the structure of this loop, by forming antibody (Figure 3). The contacts between 5F2 Fab and
hydrogen bonds with main chain oxygens of Phe30H and Tyr37H. testosterone are given in Table 2. The interface area of 5F2
In CDR-H2 (8 residues), three glycine residues (Gly–Ser–Gly–Gly) Fab in complex structure is 233 Å2, covering 37 Å2 (16%) of the
are found between the Ser57H and Ser64H and the loop was light chain and 196 Å2 (84%) of the heavy chain molecular
categorized into canonical class 3. The CDR-H3 loop of the surfaces. Thus, the heavy chain interacts predominantly with the
antibody consists of 15 amino acid residues. A salt bridge between hapten, making 76% of all atom-atom contacts. The D-ring of
Arg106H and Asp116H and a hydrogen bond between the the testosterone molecule is directed toward the bottom of the
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THE TESTOSTERONE BINDING MECHANISM

binding pocket and the amino acids interact with the A-ring and
the B-ring of the steroid skeleton. The 3-keto group oxygen of
the testosterone A-ring is close (3.3 Å) to the hydroxyl group of
the Tyr66H side chain. However, because of an unfavourable
geometry, this hydrogen bond is weak.

DISCUSSION
A 5F2 anti-testosterone antibody with a moderate testosterone
binding affinity was isolated from a naı̈ve human scFv library
(108 clones). The affinity of the 5F2 clone correlates well with
the other published examples of steroid binding antibodies
isolated from naı̈ve or designed sources of similar diversities
(Dörsam et al., 1997; Persson et al., 2006). In order to obtain an
insight into the testosterone binding mechanism of this
non-affinity maturated antibody, the crystal structure of the
5F2 Fab in complex with testosterone was determined.

Crystal structures of testosterone binding antibodies


Previously, the crystal structure of a wild-type Fab fragment from
a monoclonal anti-testosterone antibody 3-C4F5 has been
determined in free form and in complex with the testosterone
(Valjakka et al., 2002b). Also, the three-dimensional structure of
the in vitro affinity and specificity maturated form of the same
antibody, 77 Fab, has been solved (Valjakka et al., 2002a). The
mutant 77 Fab with improved affinity and specificity contains 19
Figure 2. The overall structure of 5F2 Fab in complex with testosterone.
mutations within the CDR-regions. The solved structures showed
The testosterone binding site of 5F2 Fab is a deep pocket between the
variable domains of the antibody. The light chain is shown in blue and the
that the mutations do not substantially affect the overall binding
heavy chain is in green. of testosterone, which is bound in a similar manner by both
wild-type Fab and 77 Fab. A comparison between the free and the
complexed forms of 77 Fab proved that the 77 Fab undergoes
conformational changes when it binds testosterone. A major
binding pocket, and the A-ring with the 3-keto group used for the change occurs in the conformation of the CDR-L1 loop: the tip of
conjugation with BSA is directed towards the solvent (Figures 3 the loop moves closer to the binding site in the complex
and 4). Overall, 88% of the solvent accessible area of the hapten is structure. In addition, the side-chain of the Tyr112H turns away
buried upon binding. from the binding site, thus creating an open conformation in the
Seven amino acid residues from the CDR loops of H3 and L3 as presence of testosterone. Since the attempts to crystallize 5F2
well as three framework residue from the heavy chain (Trp52H, Fab without the hapten were not successful, a free form 5F2 Fab
Ala55H, Tyr66H) form the binding site with a good shape structure is not available for the detection of possible
complementary to testosterone. Owing to the central position in hapten-induced conformational changes. However, the location
the binding site, CDR-H3 binds testosterone through five amino of two bulky hydrophobic amino acid residues (Leu110H and
acid residues: Asp107H, Tyr112aH, Tyr113H, Gly114H and Trp111H) on the solvent accessible head region of CDR-H3 may
Met115H. The light chain of 5F2 Fab is in contact with the indicate a significantly different conformation of this loop in the
testosterone through the amino acid residues Pro115L and free form structure. The crystal packing interactions illustrate that
Tyr116L from the CDR-L3 loop (Figures 3 and 4). CDR-L1, CDR-L2, the hydrophobic residues on the tip of the H3 loop are packed
CDR-H1 and CDR-H2 do not participate in the binding of the against a hydrophobic cavity of a symmetry-related 5F2 Fab
testosterone. molecule. A conformational rearrangement upon hapten binding
The interactions between the 5F2 Fab and testosterone are may explain the failure of producing free form 5F2 Fab crystals
mainly hydrophobic. In total, five aromatic amino acid residues under the same conditions as 5F2 Fab complex crystals.
surround the bound testosterone molecule, creating 70% of the
van der Waals contacts between the antibody and the hapten.
Comparison of testosterone binding between 5F2 and 77
The a-side of the testosterone is packed against Tyr116L and
Fab fragments
Tyr113H. Two other aromatic amino acids, Trp52H and Tyr112aH,
are located on opposite sides of the pocket and bind the steroid The sequence identity between the variable domains of 5F2 and
molecule in perpendicular alignment with respect to the 77 Fab is 44% for the light chain and 71% for the heavy chain. In
side-chains of residues. The b-side of testosterone with two both structures, testosterone is bound deeply into the binding
methyl groups is directed against the heavy chain of 5F2 Fab. pocket and the recognition of the testosterone is mainly based
Asp107H, Gly114H and Met115H are located on the bottom of on the hydrophobic interactions. In the structure of 77 Fab, the
the binding pocket. The side chain of Asp107H forms a hydrogen light chain of the antibody makes more contacts with the hapten:
bond with the17-hydroxyl group of the testosterone. Pro115L one amino acid from the CDR-L1 loop and four amino acids from
and Tyr66H are located near the solvent accessible surface of the the CDR-L3 loop participate in the binding of testosterone. Also,
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M. H. NIEMI ET AL.

Figure 3. The binding of testosterone to 5F2 Fab. (A) The binding of testosterone in stereo. The 5F2 Fab light chain is in blue, the heavy chain in green.
The residues interacting with testosterone are shown in light grey and testosterone in dark grey. (B) The chemical structure of testosterone and an
overview of the binding site interactions.

the CDR-H1 is in contact with testosterone through Ser35. Upon The light chain of the 5F2 Fab has only few contacts with the
hapten binding, 88 Å2 of the light chain (38%) and 146 Å2 of the testosterone molecule. Two residues are forming part of the
heavy chain (62%) solvent accessible surfaces of 77 Fab are testosterone binding site both in 5F2 and 77 Fab. Pro115L of 5F2
buried. Fab corresponds to Val114L in 77 Fab, and Tyr116L is replaced by
Three of the testosterone contacting residues of the 5F2 Fab proline in 77 Fab. However, when the side-chain of Arg32L has a
heavy chain are identical with the Fab 77 residues. Trp52H, Tyr prominent role in the formation of the binding site in 77 Fab, this
66H and Gly114H, contribute to the hapten binding in both position is occupied by the side-chain of Tyr113H in 5F2 Fab
antibodies. The positions of three contacting residues are (Figure 4).
common in 5F2 and 77 Fabs. Ala55H of 5F2 Fab corresponds 77 Fab binds testosterone in sandwich between the aromatic
to Ser55H in 77 Fab, as well as Met115H corresponds to Leu115H, amino acid residues Trp52H and Tyr112H. In the 5F2 Fab
respectively. In 5F2 Fab, Asp107H forms a hydrogen bond with structure the amino acid residues Trp52H and Tyr112aH are in
the testosterone and it is significant for the recognition of ligand the corresponding positions at the binding site. However, the
but 77 Fab has Ala107H at this position. A comparison of the orientation of the bound testosterone is different between
heavy chain contacts of 5F2 and 77 Fabs revealed also some these two testosterone binding Fab fragments. In the 5F2 Fab
differences. Both 5F2 and 77 Fab have Ser40H, but this residue is the testosterone is turned almost 908C in comparison with the
in contact with testosterone only in 77 Fab (Table 2, Figure 6). The location of testosterone in the binding site of 77 Fab (Figures 4
CDR-H3 loop is three residues longer in 5F2 compared to 77 Fab, and 5A). In 77 Fab, the a-side of testosterone packs against
in consequence, the conformation of the CDR-H3 loops are Tyr112H and the methyl groups on the b-side of testosterone
different. However, Tyr112aH of 5F2 Fab superimposes well with point toward the framework residue Trp52H, whereas in 5F2
Tyr112H of 77 Fab. Tyrosine at position 113H has a substantial Tyr112aH and Trp52H are in contact with the edges of the
effect on the shape of the binding pocket of 5F2. In Fab 77, this testosterone molecule. The different orientation of testoster-
position is occupied by a non-contacting residue valine (Figures 4 one in the binding pocket of 5F2 Fab most probably is due
and 6). to the amino acid residues of Tyr116L and Tyr113H, whose
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THE TESTOSTERONE BINDING MECHANISM

Table 2. Contacts between 5F2 Fab and testosterone

Testosterone ring Atom 5F2 Fab residue Regiona Atoms


D O17 Asp107H CDR-H3 OD2
A C1 Tyr112aH CDR-H3 CD1, CE1
C3 Tyr66H FR3 CE1, CE2, CZ, OH
C4 Pro115L CDR-L3 CB
Tyr66H FR3 CD1, CD2, CE1, CE2, CG, CZ, OH
C5 Tyr66H FR3 CD2, CE2
O3 Tyr66H FR3 CE1, CZ, OH
B C6 Pro115L CDR-L3 CB, CG
Tyr66H FR3 CB, CD2, CG
C7 Trp52H FR2 CZ2
C8 Ala55H FR2 CB
C C11 Tyr112aH CDR-H3 CD1, CD2, CE2, CG
C12 Asp107H CDR-H3 OD2
Tyr112aH CDR-H3 CB, CD2, CG
Tyr113H CDR-H3 CD2
C13 Asp107H CDR-H3 OD2
D C15 Tyr116L CDR-L3 CB, CG
Trp52H FR2 CD1, NE1
C16 Tyr116L CDR-L3 CD1, CD2, CE1, CG
Met115H CDR-H3 CE, SD
C17 Asp107H CDR-H3 OD2
Tyr113H CDR-H3 CA
O17 Asp107H CDR-H3 CB, CG
Gly114H CDR-H3 C, N, O
Met115H CDR-H3 CG
Methyl C18 Asp107H CDR-H3 CB
a
Codes refer to the IMTG definition.

location enables favourable interactions with the a-side of buried water molecule. Thus, the hydrogen bond interactions and
testosterone. especially better shape complementarity seems to explain the
The relative testosterone binding affinity of 77 Fab is higher testosterone binding capacity of 77 Fab.
approximately 3  1010 M, which is around 300-fold higher
than that of 5F2 Fab (Kd  107 M). In the 77 Fab-testosterone
Comparison to other steroid-binding antibodies
complex, one direct hydrogen bond between the main chain
oxygen of Gly114H and the 17-hydroxyl group of testosterone To date, several crystals structures of steroid binding antibodies
D-ring is formed. The 17-hydroxyl group of testosterone also have been solved. The three-dimensional structures have been
makes indirect hydrogen bonds to Ala38H and Ser40H through a determined for three anti-estradiol antibodies [57-2, 17E12 and

Figure 4. Comparison of the testosterone binding site of two antibodies. (A) The binding site of the 5F2 Fab. (B) The binding site of the 77 Fab. The
molecular surface of the light chain is in blue and the heavy chain in green. Testosterone is shown as a grey stick model.
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M. H. NIEMI ET AL.

Figure 5. Antibody–steroid complexes, superimposed with 5F2 Fab–testosterone complex (shown in grey). (A) 77/testosterone (PDB code 1vpo),
(B) 1E9dm/5b-androstane-3,17-dione (2o5z), (C) 26-10/digoxin (1igj) and (D) 10G6/estradiol-6-CMO (1jnh).

10G6 (Lamminmäki and Kankare, 2001; Monnet et al., 2002)], an mutated residue Trp52H is not in direct contact with haptens, but
anti-progesterone antibody DB3 (Arevalo et al., 1994), two it has a substantial effect on the shape of the binding pocket of
anti-digoxin antibodies (26-10 and 40-50 (Jeffrey et al., 1993; 1E9dm Fab. The depth of the binding pocket and the relative
Jeffrey et al., 1995), a glucuronide binding antibody Fv4155 position of 5b-androstane-3,17-dione with the D-ring being
fragment (Trinh et al., 1997) and a mutated catalytic Diel- deepest buried, resembles the 5F2 Fab-testosterone complex
s-Alderase antibody Fab fragment 1E9dm (Verdino et al., 2008). structure (Figure 5B). Similarly, with 5F2 Fab, the role of the
The relative orientation of the bound steroid molecule, number of antibody heavy chain in the complex formation is significant in
hydrogen bonds between the hapten and antibody and the 1E9dm-steroid structures (72 and 28% for the heavy and the light
contribution of the antibody heavy chain to steroid binding vary chain for both structures).
among these structures. By comparing the overall binding The 26-10 Fab-digoxin complex (Jeffrey et al., 1993) is another
orientation of steroid molecules three antibodies, 1E9dm, 20-10, example of a steroid binding antibody, where the heavy chain
and 10G6, show overall similarity with 5F2 Fab and were selected is responsible for the majority of the contacts between the
for more detailed comparison. In these antibody structures, the antibody and the hapten. Upon hapten binding, up to 82% of
steroid skeleton of the hapten molecule is approximately parallel the heavy chain surface buries. Two heavy chain CDR loops make
to the interface area of the variable domains (Jeffrey et al., 1995). contacts with a digoxin molecule, while the light chain of the
The anti-progesterone antibody DB3 and the catalytic antibody is in contact with the hapten through two residues from
Diels-Alderase antibody 1E9 derived from the same germ line the CDR-L3 loop. Also, in this case, the recognition of digoxin
family share a high sequence identity (91%) with distinct by Fab 26-10 is based on the surface complementarity, since
functional characters. Two mutations (Leu52HTrp and hydrogen bonds or salt bridges between the antibody and
Arg113HTrp) directed to the binding site residues of 1E9 Fab hapten are not formed. The location of a steroid skeleton of the
were sufficient to improve the steroid binding affinity of 1E9 Fab. hapten molecule is close to the testosterone in the 5F2 Fab
The crystal structures of the 1E9 double mutant Fab (1E9dm) in structure (Figure 5C). In the 26-10 Fab-digoxin complex, the
complex with progesterone and 5b-androstane-3,17-dione D-ring of the steroid molecule with the C-17 attached lactone ring
showed the major role of hydrophobic interactions in the is buried deepest in the binding pocket.
complex formation (Verdino et al., 2008). In both 1E9dm Fab The location of a bound steroid molecule is also similar in
complex structures, the hapten molecule is bound in a sandwich monoclonal anti-estradiol Fab 10G6, which has been crystallized
between the amino acid residues Trp55H and Trp113H. The in complex with estradiol-6-CMO (Monnet et al., 2002). In this
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THE TESTOSTERONE BINDING MECHANISM

Figure 6. Sequence comparison of the variable regions of heavy and light chains of 5F2 antibody with four other steroid-binding antibodies. The
residues participating in the steroid binding are shown in red and locations of the CDRs are indicated.

structure, 87% of the estradiol part of the hapten molecule is shortest CDR-H3 loops (7 and 9 residues) are found in two
buried upon binding. The D-ring with the O17-hydroxyl group is monoclonal anti-estradiol antibodies, Fab 17E12 and Fab 57-2. In
at the bottom of the binding pocket and two hydrogen bonds are these antibodies, the light chain contributes as much as the
formed between the 10G6 and estradiol-6-CMO. Three trypto- heavy chain to the hapten binding. The length of the CDR-H3
phan residues, Trp38H, Trp52H and Trp107L, surround the bound loop of the 5F2 Fab is longer, 15 residues. This is in line with the
hapten but they do not form a sandwich structure (Figure 5D). finding that human antibodies frequently have longer CDR-H3
Also, in this case, the heavy chain dominates the interactions loops than corresponding mouse antibodies (Collis et al., 2003).
between the antibody and hapten. The amino acid residues that are involved in steroid binding
The sequence comparison of the Fab region of the 5F2 can often be found in particular positions. Most of the
antibody with four other steroid binding antibodies is shown in steroid-specific antibodies are in contact with the hapten
Figure 6. The comparison reveals the importance of the CDR-H3 molecule through residues L107, L116, H40, H55, H107 and
loop, which is a commonly observed feature in antigen/hapten H115 (IMGT numbering). Another common feature in steroid
binding. binding is the utilization of aromatic residues. In particular, two
The average length of the CDR-H3 region in steroid binding positions from the heavy chain frequently carry aromatic
antibodies is 12 amino acid residues. The lengths of CDR-H3 loops side chains, 52H and 113H (Trp and Tyr in 5F2, respectively).
vary from 7 residues (Fab 17E12) to 15 residues (Fab 40-50). The The indole side-chain of tryptophan at position 113H is a part of
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M. H. NIEMI ET AL.

the sandwich structure in the binding sites of three steroid- Acknowledgements


specific antibodies, 1E9dm, 26-10 and DB3. In the case of Fab 77,
the sandwich structure is formed by the side-chains of Trp52H The skilful technical assistance of Pirkko Veijola-Bailey is greatly
and Tyr112H. Although the placing of a steroid in a sandwich appreciated. We acknowledge the European Synchrotron Radi-
is characteristic for several antibodies, not all steroid-specific ation Facility for provision of synchrotron radiation facilities and
antibodies share this binding feature. Rather, similarly with the we would like to thank the staff of beamline ID-29 for their
5F2 antibody, the hydrophobic binding site lined with aromatic assistance in data collection. This work has received financial
amino acid residues is typical for steroid binding antibodies. Thus, support from Tekes (the Finnish Funding Agency for Technology
in summary, it can be concluded that the 5F2 antibody derived and Innovation), the Academy of Finland (Finnish Centre of
from a naı̈ve human library shares the binding features that Excellence programme, 2000–2005, Project no. 64330), the Sigrid
are characteristic for anti-steroid antibodies derived from other Jusélius Foundation and the National Graduate School in Infor-
sources. mational and Structural Biology.

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