From the Departments of Protein Engineering and
¶ Protein Chemistry, Genentech, Incorporated,
South San Francisco, California 94080
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heregulins (HRGs) are epidermal growth factor
(egf) domain containing polypeptide growth factors that
bind and activate several members of the ErbB receptor family. Although
HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and
most intracellularly active receptor complexes are hetero-oligomers
containing ErbB2. The HRG egf domain was displayed on
the surface of M13 phage to facilitate mutagenic analysis and optimize
for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine
libraries were constructed in which virtually the entire sequence was
randomized in stretches of four to six amino acids. These were selected
separately for binding to immobilized ErbB3-IgG. Analysis of the
resulting sequences revealed some areas that diverged radically from
the wild-type, whereas others showed strong conservation. The degree of
wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning
mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane,
P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother,
W. J., Wells, J. A., and Sliwkowski, M. X. (1998)
J. Biol. Chem. 273, 11667-11674). Some variants from
several libraries showed significant improvements in binding affinity
to the ErbB3-IgG. These optimized segments were combined in various
ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRG
.
The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at
levels similar to wild-type. This indicates wild-type affinity is
optimized for potency and that factors other than affinity for ErbB3
are limiting. These variants showed enhanced affinity toward the ErbB4
homodimer, suggesting these receptors use very similar binding
determinants despite them having 65% sequence identity.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heregulins (HRGs)1 are growth factors that have been implicated in neuronal cell proliferation and differentiation events (1-5). They are a subset of the larger class of multidomain proteins that derive from alternate mRNA splicing of the neuregulin-1 gene, and contain an egf-like domain that by itself is sufficient for binding and activation of members of the ErbB class I tyrosine kinase receptors (1, 2, 6, 7). The ErbB receptors are overexpressed in a variety of human cancers (8), and HRGs can modulate the growth of certain cancer cell types (4, 6, 9, 10).
HRG can bind separately to ErbB3 or ErbB4 receptors, but not the ErbB2 receptor (11-13). However, ErbB2 is required for signaling and heterodimers containing ErbB2 in combination with ErbB3 or ErbB4 show the highest affinity whether tested in soluble or cell bound forms (14-18). Thus, it is believed that ErbB3 and ErbB4 contain primary binding sites for HRG and ErbB2 contributes to higher affinity either by direct interaction with the ligand or by a conformational change of the primary receptor.
The solution structure of the egf domain of HRG has
recently been determined to high resolution by NMR (19, 20). The salient features of the molecule include an N-terminal subdomain containing a central three-stranded
-sheet, a helical region, and a
smaller C-terminal subdomain that contains a short
-sheet. The
domain is stabilized by three disulfide bonds, two in the N-terminal
subdomain and one in the C-terminal subdomain (Fig. 1). Despite strong
structural similarities (20), the HRGs share limited sequence homology
with EGF (6). However, substitution of blocks of EGF sequence into HRG
did not impair binding to cells expressing ErbB3 and ErbB2 (21). HRGs
bind to dimeric ErbB receptor-IgG fusions with affinities similar to
those measured for analogous combinations expressed on the surface of
cells.2 As detailed in the
preceding paper (41), the minimal HRG
egf domain (which
differs from the
-isoform by nine substitutions near the C terminus)
has now been analyzed for ErbB3-IgG and ErbB4-IgG binding determinants
by alanine-scanning mutagenesis.
Phage display has been successfully applied to the affinity
optimization of several ligands toward a desired receptor (for review,
see Ref. 22). These include a recent report of phage display of EGF
(23). We report here the monovalent phagemid display of the
egf domain of HRG for the purpose of selecting variants
having higher affinity to the homodimeric ErbB3 receptor-IgG fusion.
The purpose was 2-fold. First, we wished to complement the
loss-of-function information of the alanine scan with a methodical optimization of stretches of sequence toward receptor binding. Given
the manageable size of the domain, this could be done comprehensively and thus reveal areas that could be functionally improved. Second, we
wanted to determine how affinity improvements for the ErbB3 receptor
would influence potency in cell-based assays and selectivity for ErbB3
versus ErbB4. In this work, we have generated HRG variants that have dramatically increased affinity to ErbB3-IgG; the recruited mutations also yield ErbB4 binding enhancements and retain wild-type like affinities to ErbB2/3 heterodimers. Our studies suggest that binding determinants on the ErbB3 and ErbB4 receptors are very similar
despite them having substantial overall sequence diversity.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Phagemids Displaying the HRG egf
Domain--
Various lengths of the HRG
egf domain gene
(residues 177-227, 177-244, 147-227, and 147-244) were amplified
from the vector pHL89 (from Dan Yansura, Genentech, Inc. (6)) by
polymerase chain reaction with primers having
NsiI/XbaI-containing overhangs. These fragments
were installed into the phagemid display vector pam-g3 (a derivative of
phGHam-g3 containing a stuffer fragment rather than the human growth
hormone gene), in which the HRG egf domain was attached to
the C terminus of pIII at residue 247, by restriction digestion and
ligation at the same sites. These generated vectors pHRG1-g3, pHRG2-g3,
pHRG4-g3, and pHRG5-g3. pHRG1-g3 was used as a template for Kunkel
mutagenesis (24) to change the A227V mutation (required by
XbaI site installation) back to wild-type to generate
pHRG6-g3. pHRG1-g3 template was also used to generate pHRG8-g3, in
which HRG177-228 (hereafter referred to as 1-52) was attached to pIII
323 through a linker containing 3 consecutive GGGS repeats, and
pHRG11-g3, in which HRG residues 1-54 were attached to pIII residue
248 through a GGGSGGG linkage. Combination mutants were constructed by
Kunkel mutagenesis, using one of the parent selectants as template.
Generation of Phagemid Libraries Displaying Randomized HRG egf Domain-- pHRG8-g3 was used as a template for constructing mutant libraries by Kunkel mutagenesis. For each library, TAA and TGA stop codons were installed at positions destined for randomization to generate custom templates that eliminated wild-type background from the pools. Positions were fully randomized by mutation to NNS codons (where N represents a mixture of all four bases and S is a mixture of G and C). One oligonucleotide was used for each library mutagenesis reaction except for library F, for which two (one randomizing positions 2 and 4, the the other 22 and 24) were used simultaneously. Mutagenesis oligonucleotides contained 18 base overhangs on either side of randomized residues. Mutagenesis reaction mixtures were electrotransformed into XL-1 blue cells (Stratagene, Inc.) and the cells were infected with 1011 plaque-forming units of KO7 helper phage. Phage stocks (~1014 phagemid/ml) were made by resuspending polyethylene glycol 8000 precipitates of culture broths from the cells after 18-24 h of growth.
Selection of Libraries for ErbB3-IgG Binding-- Wells of Nunc immunosorp 96-well plates were coated overnight with 0.5 µg of rabbit anti-human IgG (Fc-specific) antibodies (Jackson Immunoresearch) in 100 µl of 50 mM NaCO3, pH 9.6. Wells were blocked for 30 min with 200 µl of phosphate-buffered saline (0.01 M sodium phosphate, 0.1 M NaCl, pH 7.5), and 0.1% bovine serum albumin, rinsed with wash buffer (phosphate-buffered saline + 0.05% Tween 20), coated with 0.1 µg of ErbB3-IgG in binding buffer (phosphate-buffered saline + 0.1% bovine serum albumin + 0.05% Tween 20) for 1 h, and washed again. Approximately 1012 phage in 100 µl of binding buffer were applied to both the ErbB3-Ig-coated well and a control well in which no ErbB3 had been added. Following a 2-h incubation at room temperature, plates were washed extensively (12 ×) and phage eluted by treatment with 100 µl of 50 mM HCl + 0.05% Tween 20 and shaking for 10 min. Eluates were neutralized with 10 µl of 1 M Tris-Cl, pH 8.0, and 20 µl used for titration on log-phase XL-1 blue cells. The remainder was used to infect 1 ml of log-phase XL-1 blue cells (30 min at 37 °C), which were then superinfected with 2 × 1010 plaque-forming units KO7 phage and grown in 25 ml of 2YT broth containing 50 µg/ml carbenicillin for 18-24 h. Phage were harvested as described above and the cycle repeated. Following 6 rounds (libraries A, B, or D-F) or 7 rounds (libraries C or G-I) of selection, 12 clones from each were randomly sequenced by the dideoxy method (25).
Phage ELISA-- Phagemid stocks prepared from selected clones were analyzed by phage ELISA as described previously (26, 27), with slight modifications. Microtiter plates (Nunc, Maxisorp, 96 wells) were precoated with anti-IgG and coated with receptor as described for the selection procedure. For ErbB3 displacements, some assays were carried out using ErbB4-coated plates to capture uncompeted phagemids due to increased signal versus ErbB3 coat. This should not affect the measured affinities of the phagemid for soluble ErbB3 competitor so long as the receptors compete with one another for HRG-phage binding. Serial dilutions of receptor-IgG fusion and a concentration of phage predetermined to give 60% saturation (without competitor) were added to the wells in 100 µl of binding buffer. Following incubation for 2 h at room temperature, plates were washed extensively, treated with anti-M13 horseradish peroxidase conjugate (Amersham Pharmacia Biotech), and assayed for bound phagemid. EC50 values were calculated as the concentration of soluble receptor required to compete half of the phage off the plate.
Expression and Purification of Soluble HRG egf Domain Variants-- To facilitate expression of soluble egf domains, TAG codons were installed following residue 52 in the phagemids by Kunkel mutagenesis and the resulting constructs transformed into 34B8 cells. Cell cultures were grown to a density of OD550 = 1.0 in LB broth plus 50 µg/ml carbenicillin and used to inoculate modified AP5 medium (1.5 g of glucose, 11 g of Hycase SF, 0.6 g of yeast extract, 0.19 g of MgSO4, 1.07 g of NH4Cl, 3.73 g of KCl, 1.2 g of NaCl, and 0.12 mol of triethanolamine, pH 7.4, per liter), at 1/100 dilution. Cells were harvested after 24 h of growth at 30 °C (OD550 = 1.2) by centrifugation at 4500 rpm, and the pellets frozen in ethanol/dry ice for 2 h. Following resuspension and thawing in 5 mM MgCl2, 75 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, and 10 mM Tris-Cl, pH 7.6, the shocked cells were removed by centrifugation. Shockates were filtered and chromatographed by C18 reverse-phase high performance liquid chromatography using a gradient from 0 to 40% acetonitrile, and fractions shown by electrospray mass spectrometry to contain the HRG egf domain were lyophilized and resuspended in 1 mM EDTA, 10 mM Tris-Cl, pH 7.6. Proteins were found to be essentially homogeneous as determined by amino acid analysis and SDS-polyacrylamide gel electrophoresis.
Affinity Measurements and KIRA-ELISA Assays for Soluble HRG
Variants--
Receptor-IgGs were coated onto Microlite 96-well plates
as described for the selection procedure, except 1 ng (ErbB3-IgG and ErbB4-IgG) or 2.5 ng (ErbB2/3-IgG) of receptor was coated per well. A
constant and subsaturating concentration of 125I-HRG
(1-68) was incubated with varying concentrations of HRG variant in
RPMI 1640 cell culture medium (Life Technologies, Inc.) plus 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, 10 mM HEPES buffer, and 0.2% bovine serum
albumin, pH 7.2. Following incubation, wells were washed with
phosphate-buffered saline plus 0.05% Tween 20, and 100 µl of
scintillation fluid added. Plates were counted on a Hewlett-Packard
Top-count -counter. KIRA-ELISA assays were carried out as described
previously (28).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Monovalent Phage Display of the HRG egf Domain
We first constructed several M13 pIII fusions in a monovalent
format (29, 30) to determine the minimal size of the egf domain required for high-affinity receptor binding. In the initial discovery of HRG, residues 177-241 (-form) were found to bind with
similar high affinity as the full-length construct (6). Furthermore,
the data from Barbacci et al. (21), indicates that residues
177-226 of HRG
are sufficient for high-affinity binding and
activation of ErbB2/3 receptor-expressing cells. The NMR structure of
HRG
177-239 reveals that residues beyond 226 are unstructured (19,
20), and 15N relaxation data establishes that these
residues are highly flexible in
solution.3
To verify that residues immediately upstream or downstream of the
minimal egf domain were unimportant in the context of phage display, we generated HRG 147-227, 147-244, 177-227, and 177-244 as C-terminal pIII fusions. These constructs were analyzed for their
binding to the high-affinity ErbB2/3-IgG fusion by phage ELISA (Table
I) (26, 31). All bound specifically to
immobilized receptor and could be competed off with soluble receptor
with similar EC50 values (5-42 nM). The
affinities measured were ~100-fold weaker than the
Kd previously measured on cells, or the IC50 measured for radiolabeled HRG binding to ErbB 2/3-IgG
(6).2 The EC50 values obtained from phage ELISA
are sometimes higher than the true Kd, particularly
for high-affinity interactions. This may be due to the high receptor
coat concentration required to give a reasonable signal for the bound
phage, a low percentage of active receptor in competitor solutions, or
interference from the linkage of the protein to pIII (27). Clearly the
147-177 sequence bore no benefit to the HRG-phage binding, although
the constructs that extended out to 244 yielded slightly higher
affinity. To alleviate any potential steric problems occurring near the C terminus, we also tried fusions of the minimal egf domain
(177-228, hereafter referred to as 1-52) with extended flexible
linkers and fusion to pIII at residue 323 rather than 247. This yielded a mild improvement in affinity and somewhat increased functional expression as determined by titration of the phage stocks (data not
shown).
|
Phage Library Design and Selection
From the above data, construct HRG8 was chosen as a template for
designing libraries. Stretches of four to six residues at a time were
randomized in a linear fashion, except for the six cysteines,
Phe13 (which is partially buried), and the two most
C-terminal residues (Fig. 1). The
molecule was thus covered in 8 libraries. Library E, covering residues
26 to 33, contained a three-residue deletion in an effort to reduce the
size of the domain. This region corresponds to a poorly structured
-loop between the second and third
-strands of the domain, and
the equivalent amino acids are absent in EGF and transforming growth
factor
. A control construct in which residues 26-28 of HRG8 were
deleted (HRG63) showed similar ErbB3 binding as the wild-type (Table
II). An additional library (F) was
created to explore a surface patch that appeared to be important based
on the data of Barbacci et al. (21), composed of residues 2, 4, 22, and 24 from the first and second strands of the major
-sheet.
|
|
Between 1.0 × 108 and 6.4 × 108 transformants were obtained for each library, thus yielding good representation of the possible amino acid sequence combinations, particularly for libraries containing 5 or fewer randomized codons (32). Monovalent phagemids were prepared and selection performed on ErbB3-IgG fusions via capture with polyclonal antibodies to the human IgG Fc fragment. The libraries enriched rapidly, such that, by round 6, the ratio of phagemid eluted from ErbB3-IgG coated wells to anti-human Fc control wells was between 40 and 9200 (data not shown).
Selectant Sequences
The sequences obtained from random clones picked after round 6 (libraries A, B, D, E, and F) or 7 (libraries C, G, H, and I) are charted in Fig. 2A. In general there were a large number of residues that mutated to new amino acids, in some cases with dramatic changes in character. A mixture of DNA codons was found at several positions that converged to a particular residue, providing confidence that the libraries had large diversity and that selection was at the protein rather than DNA level (data not shown). In a portion of the clones in several of the libraries, a spontaneous mutation of M50I was observed. As shown below, this mutation resulted in a significant affinity enhancement for ErbB3-IgG binding. In general, the M50I-containing clones had sequences within the desired randomization window that fit the general consensus of the library. Cross-contamination between libraries occurred to a minor degree for libraries A, B, and E, but was prevalent for library H. The results are summarized below.
|
Library A-- The most striking change was the exclusive mutation S1W. Although this suggested an additional hydrophobic packing interaction with the receptor might have been recruited, this position may be more vulnerable to an expression bias (i.e. resulting in a higher level of display on phagemid) being at the point of signal peptide cleavage. His2 was mutated exclusively to hydrophilic residues. Leu3 was conserved exclusively as the wild-type residue, and Val4 came back as wild-type in 8 out of 12 clones, the remainder containing conservative substitutions with the exception of one glutamic acid. Leu3 packs against Val23 and Leu33, and Val4 packs against Met22 in the wild-type structure (20). At Lys5, two wild-type clones were found but proline dominated the position, appearing 8 times.
Library B--
This region that has helical character in the
wild-type protein showed the most dramatic changes from the natural
sequence, although in general the hydrophilic character was maintained. In particular, the six-residue stretch showed selection for glycine residues at the first and last positions (Ala7 and
Thr12), implying that the secondary structure of the area
may have completely reformed. This was also consistent with the change in register of positive and negative charges at Lys9 ( Glu, Asp, and others), Glu10 (
Arg exclusively), and
Lys11 (
Glu). Glu8 came back as many
different types of residue suggesting an absence of a specific
functional role for this side chain.
Library C--
In contrast to library B, this stretch covering the
-turn between the helix and second
-strand showed almost complete
conservation of wild-type amino acid sequence. Only two clones showed
single mutations, both at Glu19. This was consistent with
the functional importance of this region as determined by alanine
scanning mutagenesis (41). Furthermore, NMR data reveal important roles
for this turn in maintaining the structural integrity of the domain,
including several contacts to the C-terminal subdomain and positive
angles for Asn16 and Gly17 (20). In an initial
sorting experiment this library was overtaken by a contaminant
corresponding to the wild-type sequence with the M50I mutation seen in
other clones, and the sequences shown arose from a second experiment.
The vulnerability to contamination suggests an inability of this
library to enhance the affinity of the domain beyond that of the
wild-type (see library H below).
Library D--
Mutation and selection of the segment spanning
residues 21-25 primarily yielded changes in charges of two of the five
residues. Phe21 changed to tyrosine in 10 out of 12 clones,
maintaining aromaticity in the residue. Met22, which
inactivates the molecule when oxidized (20), changed to a positively
charged residue in 11 out of 12 clones. Val23 was conserved
almost exclusively, and Lys24 either remained as wild-type
or was changed to arginine, retaining the positive charge.
Asn25 mutated to -branched residues, becoming threonine
or isoleucine. The results for one particular sequence (clones D1, D2,
D8, and D12) were slightly skewed by the inclusion of the M50I
affinity-enhancing mutation, although the randomized residues selected
still agree well with the pattern found for the remaining clones.
Library E--
The sequences for the library covering the third
strand of the major -sheet were more difficult to interpret due to
the three-residue deletion included in the random mutagenesis. The
C-terminal portion of this stretch can be reasonably assumed to fall
into the wild-type
-sheet register given the adjacent
disulfide-bound Cys34, and the observation that residues
26-28 are flexible as determined by NMR.3 The first
randomized position, thus corresponding to Pro29, mutated
to threonine or tyrosine, showing a surprising isofunctionality for
these two side chains in this context. Ser30 mutated to
mixed residues with preference for a basic side chain, although glycine
also appeared twice (in sequences derived from the same clone). An
interesting switch of side chains occurred for Arg31 and
Tyr32, the first of which mutated exclusively to tyrosine
and the second primarily to arginine (7 sequences) along with leucine
(4 sequences). This was particularly unexpected given that
Tyr32 stacks with Phe13 in the structure and is
conserved in the EGF sequence (20). At Leu33, the
relatively conservative methionine mutation was found in a majority of
clones but glycine was also observed in 4 sequences.
Library F-- When His2, Val4, Met22, and Lys24 were simultaneously randomized, only one type of clone was found, in which the wild-type residues were retained except for a M22K mutation, and outside the randomized codons the M50I mutation was obtained. The M50I mutation skews the interpretation of the result since it gives the clone a selective advantage over other sequences. The selection of wild-type residues was striking, especially since His2 was not found in any of the 12 clones sequenced from library A. The neighboring Trp1 mutation in library A clones could have influenced the choice of residue at position 2, biasing it away from histidine. The Val4 retention, M22K mutation, and Lys24 retention were consistent with the selection results from the other libraries.
Library G--
Ten out of 12 clones contained a K35R mutation,
thus retaining a positive charge at this position, which lies between
two cysteines. There has been speculation that this residue is a
primary source of specificity differences between HRGs, EGF, and
transforming growth factor (33, 34). Pro37 mutated to a
mixture of hydrophilic residues, and Asn38 to a mixture of
side chains with no strong theme other than a prevalence of leucine and
valine. Glu39 came back exclusively as the wild-type
residue, and Phe40 was retained in 8 out of 12 sequences
with a conservative tyrosine mutation in 3 of the others.
Library H--
This library, comprising some of the most
functionally important residues, proved extremely vulnerable to
contamination by a high-affinity clone from library B (sequence B5).
Clone B5 was found exclusively in 12 sequences obtained after 7 rounds
of sorting versus ErbB3-IgG, and in a separate selection
experiment performed in a different laboratory with a re-made library,
it appeared again in 11 out of 12 sequences. In the one clone retaining
a sequence derived from the designed randomization of residues 41-44, a wild-type amino acid sequence was obtained except for an D43E mutation. This implies that the region requires the wild-type or very
similar sequences for optimal binding, and therefore wild-type affinities are the best that can be attained. Glu42 and
Arg44 in particular are residues that are functionally
critical (41) and are highly conserved in other EGF-like sequences.
They appear to serve important structural roles, the former by virtue
of its positive angle (in the HRG-
structure) and the latter due
to the connections provided between N- and C-terminal subdomains (hydrogen bonds between the guanidinium group and the backbone carbonyls of Thr12 and Phe13, along with
hydrophobic packing interactions with Val15 and
Phe13) (20). Since there was only one clone found that fit
the randomization scheme, the conclusions regarding the conservation of
wild-type character in this region must be treated with caution.
Library I-- Only two types of clones were found in the 12 sequenced, both having wild-type residues conserved at Gln46, Tyr48, and Val49, and both having a M50I mutation. Asn47 mutated to either histidine (9/12 clones) or tryptophan (3/12). The strong effect of M50I was witnessed by its dominance within this randomization window and the frequency of its appearance as a serendipitous mutation in clones from several other libraries.
Impact of Selected Mutations on Receptor Affinity and Specificity-- Representative clones from each library were chosen for measurement of ErbB3-IgG affinity by phage ELISA (Table II; Fig. 3). The choice of clones was based on selection frequency, with a bias toward sequences not containing the advantageous M50I substitution. Representative variants were also tested versus ErbB4 to assess specificity. The EC50 for the wild-type 1-52 phage construct for ErbB3 and ErbB4 was somewhat variable and higher (135 ± 104 and 163 ± 112 nM, respectively) than those of the free ligand as determined by 125I-HRG displacement (2.3 and 1.5 nM; Table III), and this may be due to variations in the receptor-IgG preparations used as the competitor in this format. Also, the EC50 values were higher than those determined for the HRG 177-244 construct (41), possibly due to the shorter C terminus in HRG8. However, the ratios of EC50(wt)/EC50(mutant) should be valid for assays performed with the same receptor dilutions.
|
|
Binding and Activation Properties of HRG Selectant Proteins-- An amber stop codon was installed into several phage constructs following residue 52 to facilitate periplasmic expression of soluble HRG egf domain variants. These included the wild-type 1-52, selectants A3, E2, and F1, and combinations HRG58 (D4 + E2 + M50I) and HRG72 (B3 + D4 + E2 + I2), the latter being the maximally substituted combination (containing 16 mutations total) that retained high affinity. Following purification, the variants were tested for their ability to inhibit 125I-HRG binding to ErbB2/3-, ErbB3-, and ErbB4-receptor-IgG fusions (Table III). Although the ErbB2/3-IgG also contains ErbB2 and ErbB3 homodimers (as a result of coexpression of ErbB2- and ErbB3-IgGs), the displacement of 125I-HRG should be predominantly from the ErbB2/3 receptor because of the ~100 × higher affinity of wild-type (labeled) HRG for the heterodimer versus the ErbB3 homodimer.2
The initial selectants and combinations thereof showed steep displacement plots versus the ErbB3 and ErbB4 homodimers (Fig. 4), indicating a mechanism other than simple 1:1 binding. This was most evident for the mutants displaying the highest affinity by phage ELISA. However, displacement plots for all variants versus ErbB2/3-IgG and wild-type HRG for ErbB3-IgG appeared normal for 1:1 binding. In Hill plots derived from all labeled HRG binding experiments, the wild-type 125I-HRG egf domain yielded an nH of 1.0 for ErbB3-Ig, indicative of simple 1:1 binding. The 125I-HRG58 variant gave an nH of 1.1 for ErbB2/3-IgG binding, and values of 1.4 and 1.2 were obtained for ErbB3-IgG and ErbB4-Ig, respectively (not shown), indicative of positive cooperativity. An nH value of 1.7 was recently reported for EGF binding to EGFR (39). The mutants generally showed higher affinity to the ErbB3-IgG than wild-type HRG (up to 4-fold), although the enhancements were not as strong as determined by phage ELISA, likely due to the cooperativity-related anomalous displacement behavior. As with the phage, the mutants showed similar affinity enhancements toward ErbB4-IgG as well. Mutant affinities for ErbB2/3-IgG were similar to those for wild-type HRG, although the maximally substituted construct (HRG72) bound 6-fold more weakly. It is possible that the binding affinities for the ErbB2/3-IgG are below the bottom limit of this assay format and this would mask the improved affinities. Thus, we are not willing to conclude that improvements in ErbB3 affinity do not also improve affinity for the ErbB2/3.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparison of the Selected Mutations to the Alanine Scan-- The selected HRG egf residues were classified in terms of the degree to which wild-type character is conserved. In this qualitative assessment, selection results were placed into one of four categories, as follows: 1) consensus to the wild-type amino acid; 2) substantial fraction of wild-type residue or a majority of conservative substitution (e.g. tryrosine for phenylalanine, arginine for lysine, glutamic acid for aspartic acid); 3) mutation to a mixed population of mainly non-wild-type residues; or 4) complete or nearly complete consensus to a new residue having character unlike the wild-type. These classifications are illustrated graphically on the surface plots in Fig. 6, alongside the analogous representation of the alanine-scanning mutagenesis data (41).
|
Comparison of Selected Mutations and Structurally Conserved Residues in the EGF Family of Proteins-- The sequences from 10 HRG egf domains and relatives are plotted according to frequency of occurrence in Fig. 2B. Given the differing specificities of the egf domains used in the alignment, the conservation patterns of the naturally occurring sequences and the phage-selectant residues in Fig. 2A shed light on the role of selected residues in terms of structural maintenance or binding specificity. Thus residues such as Gly18, Tyr-Phe40, Gly42, and Arg44, which are conserved across both the naturally occurring and selected sequences, are implicated as structurally important residues, and this is corroborated by the three-dimensional structural data. The conservation of other residues in the phage selection but not in the naturally occurring sequences suggested that specific roles in ErbB3 binding had been recruited. Selected residues in this category include Trp1, Gly7, Arg10, Glu11, Gly12, Arg22, Lys-Arg24, Thr25, Tyr31, Arg32, Arg35, Glu39, Gln46, Tyr48, Val49, and Ile50. Of these, Trp1, Arg35, and Glu39 did not appear to dramatically affect binding affinity when measured in the context of surrounding mutations for their particular library clone, but the remainder were implicated as affinity-enhancing mutations from the phage ELISA data.
Additivity in Combining Improved Variant Segments--
The
strategy of randomizing HRG sequentially in a blockwise fashion proved
useful in identifying areas with potential for affinity optimization,
and resulted in several variants that bind more tightly to ErbB3-IgG.
However, mutated segments were not always compatible with one another.
This can be attributed to the background within which each library
evolved and the potential for mutations from more than one library to
address a stabilizing or binding interaction that was not present in
the wild-type. For example, the Arg10 mutation was one of
several, including Lys-Arg22, Arg-Lys30,
Arg32, which would be expected to place a positive charge
in the region of the surface between the helix and the second strand of
the major -sheet (Fig. 7). This may
represent an area that makes contact with a negatively charged region
of the receptor. When two such mutations from separate libraries were
combined into the same molecule the side chains could clash and
diminish affinity, such as found for the B3 mutations when combined
with those from clone E2. Poor interactions between selectants may also
have been the cause for the poor affinities of combinations involving
the A3 sequence. These problems could be alleviated by further rounds of evolution using a "first generation" HRG combination mutant as
template, and simultaneous randomization of areas in contact with one
another.
|
Effects of Selection for ErbB3 Affinity on Receptor Specificity-- Although the HRG72 construct contained 16 mutations that were selected only by virtue of their ability to enhance ErbB3-IgG binding, the elevated affinity commuted to ErbB4-IgG binding as well. These receptors share 65% overall homology (36) and 56-67% homology in extracellular domains II-IV (37). This would suggest a strong commonality exists between receptors not only in terms of their primary HRG-binding epitopes but also the immediately surrounding areas that may be explored.
The mechanism of the HRG binding to homo- or heterodimers is still not fully understood. Recent evidence has indicated a 2:2 stoichiometry for EGF binding to the EGFR extracellular domain (38), although the question of whether the dimerization proceeds through direct contact between the ligand and both receptors (ligand-mediated) or an indirect effect that drives a receptor conformational change (receptor-mediated) cannot be resolved. The Hill plot slopes of >1 observed for EGF binding to EGFR (39) and for affinity-optimized HRG variants and ErbB3-IgG or ErbB4-IgG suggests a 2:2 stoichiometry also exists for the latter receptors. If a 2:2 binding mechanism is operational for ErbB3-IgG, the cooperativity could be enhanced by the selected mutations if, for the first ligand bound, they assist in setting up the complex for higher affinity binding to the second ligand. This could occur by either direct bridging of the receptor pair or by conformational change. The cooperativity observed for the optimized mutants makes it difficult to compare the absolute binding affinity to wild-type HRG for ErbB3-IgG and ErbB4-IgG. Recently, Tzahar et al. (40) have reported a very low affinity constant (800 nM) for binding of ErbB2-IgG to HRG. Interestingly, we observe that cooperativity was not as prevalent for binding to ErbB2/3-IgG. Taken together these data suggest a stoichiometry of 1 ligand:2 receptors in the heterodimer. ErbB2/3 binding and signaling was not significantly impaired or enhanced by optimization of ErbB3 binding. This suggested that the enhancement of affinity seen in ErbB2/3 heteromeric complexes was not strongly dependent on the HRG sequence outside of the primary ErbB3 site. Receptor selective HRG variants could potentially be generated by incorporating negative selection into the phagemid panning procedure. Receptor selective variants may be of therapeutic value if they exert a dominant-negative phenotype, e.g. if they could bind ErbB3 but not activate ErbB2/3 complexes. They may also have utility in dissecting out the specific signaling pathways utilized by each ErbB receptor subtype. ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Mike Sadick for KIRA-ELISA assays, and Andrew Braisted and Paul Pisacane for technical assistance.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by an National Research Service Award Fellowship from National Institutes of Heath Postdoctoral Training Grant GM16549-01. Present address: Chiron Corp., Dept. of Biological Chemistry, 4560 Horton St., Emeryville, CA 94608.
To whom correspondence should be addressed: Genentech Inc., MS
27, South San Francisco, CA 94080. Tel.: 650-225-1177; Fax: 650-225-3734; E-mail: jaw{at}gene.com.
1 The abbreviations used are: HRG, heregulin; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay.
2 V. D. Fitzpatrick, P. I. Pisacane, R. L. Vandlen, and M. X. Sliwkowski, manuscript in preparation.
3 Fairbrother, W. J., Liu, J., Pisacane, P. I., Sliwkowski, M. X., and Palmer, A. G., III (1998) J. Mol. Biol., in press.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|