Molecular Recognition by a Binary Code - ResearchGate

doi:10.1016/j.jmb.2005.03.041 J. Mol. Biol. (2005) 348, 1153–1162
Molecular Recognition by a Binary Code
Frederic A. Fellouse, Bing Li, Deanne M. Compaan, Andrew A. Peden
Sarah G. Hymowitz and Sachdev S. Sidhu*
Department of Protein
Engineering, Genentech Inc
1 DNA Way, South San
Francisco, CA 94080, USA
*Corresponding author
Functional antibodies were obtained from a library of antigen-binding sites
generated by a binary code restricted to tyrosine and serine. An antibody
raised against human vascular endothelial growth factor recognized the
antigen with high affinity (K D Z60 nM) and high specificity in cell-based
assays. The crystal structure of another antigen binding fragment in
complex with its antigen (human death receptor DR5) revealed the
structural basis for this minimalist mode of molecular recognition. Natural
antigen-binding sites are enriched for tyrosine and serine, and we show
that these amino acid residues are intrinsically well suited for molecular
recognition. Furthermore, these results demonstrate that molecular
recognition can evolve from even the simplest chemical diversity.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: phage display; protein engineering; combinatorial mutagenesis;
antibody library; vascular endothelial growth factor
Introduction
The natural immune repertoire can generate
antibodies that recognize essentially any antigen
with high specificity and affinity. Antigen recognition
is mediated by six complementarity determining
regions (CDRs) 1,2 that present a large
surface for contact with antigen. 3,4 CDR sequences
are hypervariable, but it has become clear that the
overall composition of functional CDRs is biased in
favor of certain amino acid types. 5–11 In particular,
tyrosine and serine residues are highly abundant,
and tyrosine residues are involved in a disproportionate
number of antigen contacts. 12–14
The overabundance of tyrosine and serine
residues may be a coincidental consequence of
biases that exist in the sequences of antibody
genes. 5,15–18 Alternatively, it is possible that these
amino acid residues are particularly well suited for
molecular recognition, and as a result, the immune
system has evolved under selective pressure for the
Abbreviations used: CDR, complementarity
determining region; CDR-Hn (where nZ1,2 or 3), heavy
chain CDR 1,2 or 3; CDR-L3, light chain CDR3; ECD,
extracellular domain; ELISA, enzyme-linked
immunosorbent assay; Fab, antigen-binding fragment;
hDR5, human death receptor DR5; hVEGF, human
vascular endothelial growth factor; GFP, green fluorescent
protein.
E-mail address of the corresponding author:
sidhu@gene.com
enrichment of tyrosine and serine in functional
antigen-binding sites. 5,12,19,20
We previously addressed this question by generating
synthetic antibody libraries with solvent
accessible CDR positions randomized with a
degenerate codon that encodes for only four
amino acid residues (tyrosine, serine, alanine and
aspartate). 20 Surprisingly, we were able to generate
high affinity antibodies against human vascular
endothelial growth factor (hVEGF), an angiogenic
hormone that has been implicated in tumorogenesis.
21,22 Structural analyses suggested that it
may be possible to simplify the code for antigen
recognition even further, as most of the contacts
with antigen were mediated by tyrosine sidechains,
while aspartate was almost entirely
excluded from the binding sites.
We now show that a binary code, restricted to
only tyrosine and serine, is sufficient to mediate
antigen recognition with high affinity and
specificity. Thus, it appears that these amino acid
residues are particularly well suited for molecular
recognition, and furthermore, naïve antigen recognition
can evolve from even the simplest chemical
diversity.
Results
Antibodies from a binary code
We constructed two libraries of phage-displayed
antigen-binding fragments (Fabs) by randomizing
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

1154 Molecular Recognition by a Binary Code
CDR positions within a humanized Fab framework
with a binary degenerate codon that encodes for
equal proportions of tyrosine and serine. Solventaccessible
positions in the first and second CDRs of
the heavy chain (CDR-H1 and CDR-H2) were
randomized, and random loops of variable lengths
were inserted in place of seven residues in the third
heavy chain CDR (CDR-H3). The libraries differed
in that library A had a fixed light chain while the
third CDR of the light chain (CDR-L3) was
randomized in library B. Each library contained
w10 10 unique members, and thus, the actual library
diversities were comparable to the maximum
number of unique sequences encoded by each
library design (w4!10 9 ).
The libraries were used to generate Fabs against a
panel of six protein antigens. Specific binding
clones were identified by phage enzyme-linked
immunosorbent assay (ELISA) screening, and were
defined as Fab-phage that bound to the cognate
antigen but did not exhibit detectable binding to
seven other proteins. Specific Fabs were isolated
against all six antigens and DNA sequencing
revealed diverse CDR sequences that contained
only tyrosine and serine at the randomized sites
(Figure 1). Competitive phage ELISAs were used to
estimate affinities as IC 50 values. Fabs exhibited
high affinity for four of the antigens (IC 50 !100 nM),
moderate affinity for insulin (IC 50 Z1.4 mM) and low
affinity for maltose binding protein (IC 50 O5 mM).
Thus, the binary chemical diversity was sufficient to
mediate specific recognition of all six antigens, and
for four of the antigens the binding interactions
were of high affinity. Fabs against two of the
antigens (hVEGF and human death receptor DR5
(hDR5)) were chosen for further functional and
structural characterization.
Functional characterization of anti-hVEGF Fabs
DNA sequencing of 184 hVEGF-binding clones
revealed 63 unique sequences, and some of these
are shown in Figure 2(a). Most of the clones were
from library B but some clones were also isolated
from library A. Interestingly, the clones from library
B exhibit homology within the selected CDR-L3 and
CDR-H3 sequences. In contrast, the CDR-H3
sequences from library A exhibit homology
amongst themselves but are very different from
the sequences from library B. Thus, it appears that
the nature of the CDR-L3 sequence influenced the
selection of CDR-H3 sequences, and as a result, two
distinct classes of anti-hVEGF antibodies arose from
the different libraries. The clones were screened by
competitive phage ELISA and exhibited IC 50 values
Figure 1. Sequences and specificity profiles of synthetic Fabs. (a) CDR sequences in the randomized regions. The CDR-
L3 sequence is not shown for Fabs selected from library A in which the light chain was not randomized. Tyrosine and
serine residues are shown in yellow or red, respectively. Residues in grey are buried residues that were not randomized
in the libraries. The numbering is according to the nomenclature of Kabat et al. 1 (b) Fab-phage selected for binding to a
particular antigen (cognate antigen, x-axis) were assayed for binding to various immobilized proteins by phage ELISA
and bound phage were detected spectrophotometrically (optical density at 450 nm, y-axis). Affinities for cognate antigen
were estimated as IC 50 values by competitive phage ELISA. 37,38 The following proteins were used: neutravidin (NAV, red
bar), maltose binding protein (MBP, orange bar), Erbin PDZ domain (PDZ, yellow bar), insulin (green bar), human
vascular endothelial growth factor (hVEGF, blue bar), human death receptor 5 (hDR5, pink bar), glutathione-Stransferase
(purple bar), human immunoglobulin G (black bar), bovine serum albumin (white bar).

Molecular Recognition by a Binary Code 1155
Figure 2. Affinity and specificity
of anti-hVEGF Fabs. (a) CDR
sequences of anti-hVEGF Fabs.
The numbering is according to the
nomenclature of Kabat et al. 1 and
only positions that were randomized
in the libraries are shown.
Clones 1 through 7 were isolated
from library B, while clones 8
through 14 were isolated from
library A. Dashes indicate gaps in
the alignment. (b) Kinetic analysis
of Fabs binding to immobilized
hVEGF. The surface plasmon
resonance traces are shown for
Fab-YSv1 at four different concentrations
(63 nM, 125 nM, 250 nM
and 500 nM). Experimental data
are represented by open circles
and the global fits are represented
by continuous lines. Kinetic parameters
were determined for Fabs
YSv1, YSv2 and YSv3. (c) Immunofluorescence
staining with Fab-
YSv1 (red) performed on A673
cells expressing murine VEGF-
GFP (green). VEGF-GFP and Fab-
YSv1 staining co-localize in the
extracellular space formed between
cell-to-cell contacts (merge,
yellow). The Fab-YSv1 staining
was completely abolished by
pre-incubation of the antibody
with excess recombinant hVEGF
(C VEGF panels). The scale bar
represents 20 mm. (d) Immunoprecipitations
performed on media
collected from metabolically
labeled A673 cells. Fab-YSv1 and
monoclonal antibody A4.6.1 show
comparable specificity, as evidenced
by identical patterns of
bands for precipitated hVEGF isoforms.
Anti-GFP polyclonal antibody
was used as a negative
control.
ranging from approximately 50 nM to greater than
5 mM.
The three anti-hVEGF clones with the highest
estimated affinities (top three sequences in
Figure 2(a)) were purified as free Fab proteins.
The binding kinetics of the purified Fabs
(designated YSv1, YSv2 and YSv3) were studied
by surface plasmon resonance (Figure 2(b)).
Fab-YSv1 exhibited the highest affinity for hVEGF
(K d Z60 nM), while the other two Fabs bound
approximately fivefold less tightly due to faster off
rates. It is notable that the sequences of Fab-YSv1
and Fab-YSv2 differ in only three positions, and
thus, these three differences account for the
improved affinity of Fab-YSv1 in comparison with
Fab-YSv2.
We next investigated the specificity of Fab-YSv1
by using the protein to visualize VEGF in
mammalian cells transfected with a gene encoding
for VEGF fused to green fluorescent protein (GFP).
The immunohistochemical staining with Fab-YSv1
precisely overlapped with the fluorescence signal
from the VEGF-GFP fusion (Figure 2(c)). Furthermore,
the signal was completely blocked by
incubating Fab-YSv1 with hVEGF prior to staining.
We also conducted immunoprecipitations of
endogenous hVEGF and compared the performance
of Fab-YSv1 to that of a highly specific, natural
anti-hVEGF monoclonal antibody (A4.6.1). 23 Both
antibodies immunoprecipitated an identical set of
bands (Figure 2(d)) that likely represent hVEGF
variants generated by alternative mRNA splicing. 23
Taken together, these results show that Fab-YSv1
binds to hVEGF with high affinity and specificity
comparable to that of a natural antibody, even in the
complex cellular milieu.

1156 Molecular Recognition by a Binary Code
Structural characterization of an anti-hDR5 Fab
To gain insights into the structural basis for
antigen recognition by the binary code, we studied
a Fab that was selected for binding to the
extracellular domain of hDR5 (hDR5-ECD), a cellsurface
receptor that mediates apoptotic cell
death. 24 Nine unique anti-hDR5 clones were
obtained, and the Fab that exhibited the highest
estimated affinity by competitive phage ELISA
(YSd1; Figure 1(a)) also bound to hDR5 with high
affinity in surface plasmon resonance experiments
(K d Z34 nM).
The crystal structure of Fab-YSd1 in complex
with the hDR5-ECD was solved and refined to
3.35 Å resolution (Figure 3(a) and Table 1). While
the structure is only of modest resolution, the
quality of the structure is quite good with 98.9%
Table 1. Data collection and refinement statistics for the
Fab-YSd1:hDR5-ECD complex
A. Diffraction data
Space group P3 2 21
Unit cell constants (Å)
aZ147, cZ145
Resolution (Å)
50–3.35 (3.47–3.35) a
b
R sym 0.12 (0.34) a
hI/sIi
8.3 (4.5) a
No. of reflections 149,117
No. of unique reflections 26,539
Completeness (%)
99.7 (99.8) a
B. Refinement
Resolution (Å) 30–3.35
No. of reflections 26,003
c
R work , R free 0.224, 0.280 (0.232, 0.281)
No. of residues 1056
No. of non-H atoms 8,026
rmsd bond length (Å) 0.012
rmsd angles (deg.) 1.3
rmsd B (bonded atoms) (Å 2 ) 2.3
Ramachandran plot (%) d 83.4, 15.5, 0.7, 0.4
a Numbers in parentheses refer to the highest resolution shell.
b R sym ZSjIKhIij=SI, where hIi is the average intensity of
symmetry related observations of a unique reflection.
c R work ZSjF o KF c j=SF o , where F o and F c are the observed and
calculated structure factor amplitudes, respectively. R free is the
R-factor for a randomly selected 10% of the reflections excluded
from all refinement.
d Percentage of residues in the most favored, additionally
allowed, generously allowed, and disallowed regions of a
Ramachandran plot.
Figure 3. The structure of Fab-YSd1 bound to the hDR5-
ECD. (a) The complex of the hDR5-ECD with Fab-YSd1.
The hDR5-ECD is depicted as a white molecular surface.
The main-chain of Fab-YSd1 is shown as sticks; the light
and heavy chains are shown in green and blue,
respectively, and CDR-H3 is shown in yellow.
(b) Experimental density for CDR-H3. The F o KF c omit
map contoured at 2.75s shows unbiased electron density
(blue) for CDR-H3. Phase bias was removed by omitting
CDR-H3 prior to refinement. The coordinates of the final
refined model for CDR-H3 are shown as sticks, colored
according to atom type (carbon, yellow; oxygen, red;
nitrogen, blue), with side-chains rendered as sticks. The
hDR5-ECD is shown as a white molecular surface.
Despite the relatively modest resolution of the structure
(3.35 Å), the electron density maps are of high quality and
allow for accurate determination of backbone and sidechain
positions.
of residues in the most favored or additionally
allowed parts of the Ramachandran plot. The data
quality was limited by the availability of single
crystals. All crystals showed evidence of more than
one lattice, and thus, to collect a high quality data
set, it was necessary to screen many crystals and to
then find the most single portion of the best crystal.
Nonetheless, the maps were of excellent clarity and
the initial maps calculated with phases from the
molecular replacement solution showed continuous
density for CDR-H3 with side-chain density for
many of the tyrosine residues (Figure 3(b)). The
presence of two copies of the Fab:antigen complex
in the asymmetric unit permitted the use of NCS
restraints which aided the refinement process.
Additionally, tyrosine is a large side-chain with a
distinctive shape, and thus, it can be positioned
accurately even in maps at a resolution of 3.35 Å.
Other than the unusual CDR-H3 conformation,
the structure of Fab-YSd1 is similar to that of the
humanized Fab-4D5 variant that was used as the
scaffold for the antibody library 20,25 (Figure 4(a)),
although the elbow angles differ due to differences
in crystal packing. Overall, the structure shows that
Fab-YSd1 interacts with antigen in a normal
manner, but tyrosine residues form most of the
contact surface (Figure 4(b) and (c)). Antigen
binding results in the burial of 460 Å 2 on the
heavy chain and 270 Å 2 on the light chain. Tyrosine
and serine residues within CDRs H2, H3 and L3
account for 58% and 21% of the buried surface area,
respectively. The remainder of the buried surface
area (21%) is contributed by CDR-L1, which was not
randomized and lies on the periphery of the

Molecular Recognition by a Binary Code 1157
interface (Figure 4(b)). On the antigen side of the
interface, the buried surface area is contributed by a
variety of amino acid types, but aromatic residues
only account for a small fraction (14%) of the
structural epitope. Thus, tyrosine plays a dominant
role in antigen recognition by Fab-YSd1, but it does
so by utilizing interactions with a diverse array of
functional groups on the antigen, and interactions
with aromatic side-chains do not play a major role.
The very long CDR-H3 of Fab-YSd1 protrudes
from the framework and makes extensive contact
with hDR5, contributing 39% of the surface area
buried upon antigen binding (288 Å 2 ). Structures of
antibodies with long CDR-H3 loops have been
reported, 26–28 but Fab-YSd1 is unique in that CDR-
H3 contains a “biphasic” helix with tyrosine and
serine residues clustered on opposite faces
(Figure 4(d)). The tyrosine face is buried against
the surface of hDR5, and as a result, 99% of the
CDR-H3 accessible surface buried upon binding to
hDR5 is contributed by tyrosine, and serine
residues likely play structural roles. In this regard,
CDR-H3 exemplifies how even the most minimalist
binary diversity can give rise to a structural solution
that mediates antigen recognition with high affinity
and specificity.
Fab-YSd1 binds to the N-terminal portion of
hDR5 and occludes the smaller of two binding sites
for the natural hDR5 ligand Apo2L/TRAIL 29
(Figure 5(a)). Intriguingly, one of the Apo2L/
TRAIL residues that contributes the most binding
energy to the interaction with hDR5 is Tyr216, as
substitution of this residue by alanine causes a 320-
fold loss in biological activity and a ninefold loss in
affinity for hDR5. 30 Tyr216 binds in a hydrophobic
pocket in cysteine-rich domain 2 of hDR5 29
(Figure 5(b)), resulting in a snug fit that buries
greater than 90% of the side-chain. Although Fab-
YSd1 uses many tyrosine residues to bind to hDR5,
none of them precisely mimics this interaction.
Tyr56 of the heavy chain occupies a similar position
as Tyr216 of Apo2L/TRAIL, but it lies along the
antigen surface and only 60% of the side-chain is
buried (Figure 5(b)).
This structure is also the first for a tissue necrosis
factor receptor (TNFR) family member bound to an
antibody fragment, and as such, it provides insight
into how an antibody recognizes antigens of this
type. Overall, the structure of hDR5 bound to Fab-
YSd1 is similar to that of hDR5 bound to Apo2L/
TRAIL. 29 Cysteine-rich domains 1 and 2 (residues
22–86) from both structures superimpose on each
other with a pair-wise root-mean-square deviation
(rmsd) of less than 0.30 Å 2 on C a atoms. This is the
same level of structural conservation observed
amongst independent structures of hDR5 bound
to Apo2L/TRAIL. 29 As in the Apo2L:hDR5
complex structure, which contains three crystallographically
independent copies of hDR5, the
orientation of cysteine-rich domain 3 in the two
independent copies of hDR5 bound to Fab-YSd1
varies somewhat with respect to cysteine-rich
domains 1 and 2, although variations between the
hDR5 molecules in the Fab-YSd1 and Apo2L/
TRAIL complexes are more extreme than those
within either group. Thus, in both complexes, it
appears that cysteine-rich domains 1 and 2 behave
as a rigid body, while in contrast the third domain
exhibits flexibility with respect to the rest of hDR5.
Cysteine-rich domain 3 does not make contact
with Fab-YSd1, but it makes extensive contact with
Apo2L/TRAIL (Figure 5(a)). In both copies of the
Fab:antigen complex contained within the asymmetric
unit, cysteine-rich domain 3 is less well
defined than it is in the structure of hDR5 bound
to Apo2L/TRAIL. In particular, residues 91–104
exhibit poor density in the complex with Fab-YSd1,
but this region is well-ordered when bound to
Apo2L/TRAIL, and taken together, these differences
suggest that the loop is not well-ordered in
the absence of Apo2L/TRAIL. The membrane
proximal end of cysteine-rich domain 3, including
the last disulfide, is also poorly ordered in the
complex with Fab-YSd1. Disorder is not uncommon
in crystal structures of members of the TNFR family,
as for example, the entire final cysteine-rich domain
of TNFR1 is disordered in the complex with
lymphotoxin. 31
Discussion
Despite extreme restrictions on chemical diversity,
we isolated synthetic Fabs with affinities
comparable to those obtained from naïve libraries
designed with high chemical diversity. Furthermore,
the affinities compare favorably with the
affinities of antibodies from the naïve immune
response, which are often in the micromolar range
prior to affinity maturation through somatic
mutation mechanisms. 16 Our results provide
insight into how the naïve immune repertoire can
evolve high affinity antibodies against a seemingly
endless array of antigens. It appears that the
selection of antibodies with affinities and specificities
sufficient for initial selection from a naïve pool
is a relatively simple process that may require only
a few productive binding contacts. Thus, the limited
diversity of the naïve immune repertoire 32 is
sufficient to generate initial binding clones that
can then attain improved function through subsequent
cycles of affinity maturation.
We focused our study on protein antigens, but it
will be interesting to see if the limited diversity
approach will be successful against other antigen
classes (e.g. small molecules or carbohydrates).
With the exception of CDR-H3, we also restricted
CDR conformational diversity, as we did not
randomize buried residues which influence CDR
main-chain conformations. 33 Thus, it may be
possible to obtain further improvements in binding
by targeting these regions and framework positions
that are known to modulate affinity. 25,34 The binary
binding surfaces represent a minimal benchmark
for antigen recognition into which additional
diversity and conformational flexibility can be

1158 Molecular Recognition by a Binary Code
Figure 4 (legend opposite)

Molecular Recognition by a Binary Code 1159
introduced to study the details of affinity maturation.
In particular, these systems may be very
amenable to computer modeling studies, since both
serine and tyrosine have few rotamers, and the
simplicity of the system should simplify the
computational process.
Our results suggest that the high abundance of
tyrosine and serine in antibody combining sites
reflects an innate capacity for these amino acid
residues to mediate antigen recognition. Both
residues are hydrophilic and thus can be exposed
on surfaces without promoting non-specific aggregation,
but at the same time, both can also be
accommodated readily at protein–protein interfaces.
Because of its small size and neutral nature,
the serine side-chain is unlikely to cause unfavorable
steric hindrance or electrostatic repulsions. On
the other hand, the large tyrosine side-chain
contributes substantial surface area for contact,
and yet, it has a unique chemical nature which
allows it to participate in favorable binding interactions
with many different types of functional
groups. 5,9,12,20 We hypothesize that the large tyrosine
side-chain acts as a functional group that
makes key binding contacts with antigen, while the
small serine side-chain serves as an auxiliary group
that provides space and flexibility for tyrosine to
establish optimal binding contacts. Due to their
complementary nature, it appears that tyrosine and
serine may be particularly well suited to work
together in antigen recognition.
Much effort has been expended on the development
of large, highly diverse libraries that can be
used to obtain antagonists and agonists of biological
processes. 35–38 However, our results demonstrate
that it is possible, and perhaps even advantageous,
to restrict chemical diversity to small subsets of
functional groups that are particularly well suited
for mediating molecular recognition.
Materials and Methods
Library construction and sorting
A phagemid designed to display bivalent, humanized
Fab-4D5 on the surface of M13 bacteriophage was used to
construct libraries, as described. 20,39 Oligonucleotidedirected
mutagenesis was used to replace CDR positions
with TMT degenerate codons, (MZA/C in equal proportions).
The positions chosen for randomization in
CDR-L3, CDR-H1 and CDR-H2 are shown in Figure 1. In
CDR-H3, positions 95 through 100a were replaced with
random loops of all possible lengths ranging from 7 to 20
residues (library A) or 7 to 15 residues (library B).
Phage from the libraries were cycled through rounds of
binding selection with antigen immobilized on 96 well
Maxisorp immunoplates (NUNC) as the capture target, as
described. After five rounds of selection, phage were
produced from individual clones grown in a 96 well
format and the culture supernatants were used in phage
ELISAs to detect specific binding clones.
Competitive phage ELISA
A modified phage ELISA 37,38 was used to estimate the
binding affinities of Fabs. Phage ELISAs were carried out
on plates coated with antigen, as described above. Phage
displaying antibody fragments were serially diluted in
phosphate-buffered saline (PBS), 0.5% (w/v) bovine
serum albumin (BSA), 0.1% (v/v) Tween 20, and binding
was measured to determine a phage concentration giving
w50% of the signal at saturation. A fixed, sub-saturating
concentration of phage was pre-incubated for two hours
with serial dilutions of antigen and then transferred to
assay plates coated with antigen. After 15 minutes
incubation, the plates were washed with PBS, 0.05%
Tween 20 and incubated 30 minutes with horseradish
peroxidase/anti-M13 antibody conjugate (1:5000
dilution) (Pharmacia). The plates were washed,
developed with TMB substrate (Kirkegaard and Perry
Laboratories), quenched with 1.0 M H 3 PO 4 , and read
spectrophotometrically at 450 nm. The binding affinities
of were estimated as IC 50 values defined as the
concentration of antigen that blocked 50% of the phage
binding to the immobilized antigen.
Protein purification and affinity analysis
Fab proteins were purified from Escherichia coli as
described. 40 Binding kinetics were determined by surface
plasmon resonance using a BIAcoree-3000 with hVEGF
immobilized on CM5 chips at w500 response units, as
described. 41 Serial dilutions of Fab proteins were injected,
and binding responses were corrected by subtraction of
responses on a blank flow cell. For kinetic analysis, a 1:1
Langmuir model of global fittings of k on and k off was used.
Figure 4. Antigen binding site of Fab-YSd1 and the binding interface with hDR5-ECD. (a) Stereo view of the
superimposition of the variable domains of Fab-YSd1 and humanized Fab-4D5 (1N8Z). 46 The Fab variable domains are
represented as a cartoon. CDR-H3 of Fab-YSd1 is colored yellow; the rest of the Fab-YSd1 heavy chain is in blue and the
light chain is in green. Humanized Fab-4D5 heavy and light chains are colored cyan and red, respectively. (b) Stereo view
of CDR loops that contain residues in contact with the hDR5-ECD (closer than 4.5 Å). The CDR loops are labeled and are
shown as transparent cartoons. CDR side-chains that make contact with the hDR5-ECD are rendered as sticks and are
colored as follows: tyrosine, blue; serine, green; all other amino acids, white. A fragment of the hDR5-ECD is shown as a
molecular surface; residues in contact with the heavy or light chains are colored yellow or red, respectively, and residues
in contact with both are colored orange. (c) The amino acid composition of the interface between Fab-YSd1 and the
hDR5-ECD. Each circle represents the surface area on Fab-YSd1 or the hDR5-ECD that becomes buried upon complex
formation. The colors indicate the proportion of the buried surface area contributed by each amino acid type, and all
amino acid types that contribute more than 1% of the total buried surface area on each molecule are shown. (d) Contacts
between CDR-H3 and the hDR5-ECD. The hDR5-ECD is shown as a molecular surface and residues that contact CDR-
H3 are colored pink. The CDR-H3 main-chain is depicted as a yellow cartoon; tyrosine and serine side-chains are
rendered as sticks and colored blue or green, respectively.

1160 Molecular Recognition by a Binary Code
The K d values were determined from the ratios of k on and
k off .
Immunohistochemistry
Human A673 cells expressing murine VEGF-GFP were
stained and imaged, as described. 42 In the plus VEGF
panel, Fab-YSv1 was pre-incubated for five minutes with
a fivefold excess of recombinant hVEGF before being
incubated with the cells.
Immunoprecipitation
A673 cells were metabolically labeled and immunoprecipitations
were performed from the media, as
described, 23 using 15 mg of anti-GFP polyclonal antibody
(Clontech), Fab-YSv1 or monoclonal antibody A4.6.1. The
immune complexes were eluted by boiling and resolved
by SDS-PAGE on a 14% (w/v) acrylamide gel under
reducing conditions. The gel was dried and then exposed
to a phosphoimager plate overnight.
Crystallization, structure determination and
refinement
DNA coding for the hDR5-ECD (residues 1–130) with
an N-terminal His-tag was cloned into the baculovirus
transfer vector pAcGP67-B (Pharmingen). Following
transfection and viral amplification in Sf9 cells, protein
was expressed and secreted from Hi5 cells grown at 27 8C.
The culture medium was supplemented with 50 mM Tris
(pH 8.0), 1 mM NiCl 2 , 5 mM CaCl 2 , and 1 mM phenylmethylsulfonyl
fluoride. The pH was adjusted to pH 7.6
and the medium was filtered prior to loading on a Ni-
NTA column (Qiagen). After elution with imidazole, the
His-tag was removed by digestion with thrombin
followed by purification on a Superdex-75 sizing column.
Fab-YSd1 was expressed in E. coli and purified over a
Protein G column. 40 Fab-containing fractions were further
purified by passage over a S-Sepharose column, followed
by a Superdex-200 sizing column.
Fab–receptor complex was made by mixing Fab-YSd1
with an excess of the hDR5-ECD domain, followed by
purification over a Superdex-200 sizing column. The
complex-containing fractions were pooled and concentrated
to 11 mg/ml in 150 mM NaCl, 20 mM bis-Tris
(pH 6.5) and used for crystallization trials. Crystals grew
at 19 8C from hanging drops containing an equal volume
of protein and well solution consisting of 20% polyethylene
glycol (PEG) 8000, 0.2 M magnesium acetate,
0.1 M sodium cacodylate (pH 6.2–6.6). Crystals were
transferred to mother liquor containing the well solution
with 20% PEG 300, prior to cryo-cooling by immersion in
liquid nitrogen. The resulting crystals were visibly nonsingle
and it was necessary to screen many crystals to find
one that was mostly single.
A 3.35 Å data set was collected at beamline 19BM at the
APS. Data processing was done with HKL. 43 Calculation
of the Matthew’s coefficient indicated that the asymmetric
Figure 5. Comparison of the interactions of hDR5 with
Fab-YSd1 and Apo2L/TRAIL. (a) Mapping of the
structural binding epitopes of hDR5 for Fab-YSd1 or
Apo2L/TRAIL. The hDR5-ECD is shown as a molecular
surface. The unshared portions of the epitopes for
binding to Fab-YSd1 or Apo2L/TRAIL are colored yellow
or blue, respectively, and the shared portion is in green.
(b) Comparison of Tyr56 of Fab-YSd1 and Tyr216 of
Apo2L/TRAIL. The hDR5-ECD is shown as a molecular
surface and colored as in (a). Tyr56 of Fab-YSd1 and
Tyr216 of Apo2L/TRAIL are rendered as sticks and
colored cyan or pink, respectively, as are the a-carbon
traces of the surrounding loops. Structures were drawn
with PyMOL (DeLano Scientific, San Carlos, CA).

Molecular Recognition by a Binary Code 1161
unit of the crystals contained two Fab:antigen complexes.
The structure was solved in space group P3 2 21 by
molecular replacement with the program Phaser 44 using
the structures of two Fab-4D5 variants as search models. 25
The resulting maps showed clear density for the hDR5-
ECD, which was placed manually, as well as for the Fab
CDRs. Refinement was performed with the program
REFMAC5 45 and included TLS refinement and NCS
restraints imposed separately on the variable and
constant domains of Fab-YSd1, as well as on the hDR5-
ECD.
Protein Data Bank accession numbers
The coordinates and structure factors for the Fab-
YSd1:hDR5-ECD complex have been deposited in the
RCSB Protein Data Bank (PDB:1ZA3).
Acknowledgements
We are grateful to the Genentech Fermentation,
DNA Synthesis and DNA Sequencing groups. We
thank M. Franklin, A. Cochran, A. de Vos and
H. Lowman for helpful discussions and H. Darbon
for his support. We also thank Stephan L. Ginell,
PhD of beamline 19BM at the APS (Advanced
Photon Source). Use of the Argonne National
Laboratory Structural Biology Center beamlines at
the Advanced Photon Source, was supported by the
US Department of Energy, Office of Energy
Research, under contract no. W-31-109-ENG-38.
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Edited by I. Wilson
(Received 22 December 2004; received in revised form 10 March 2005; accepted 14 March 2005)