(Received for publication, February 14, 1995; and in revised form, April 25, 1995)
From the
The crystal structure of the complex between the cross-reacting
antigen Guinea fowl lysozyme and the Fab from monoclonal antibody
F9.13.7, raised against hen egg lysozyme, has been determined by x-ray
diffraction to 3-Å resolution. The antibody interacts with
exposed residues of an Antibody molecules convey a potentially unlimited molecular
diversity and antigen-binding repertoire. Although the genetic elements
and mechanisms underlying the generation of this functional and
structural diversity have been characterized (Tonegawa, 1983), an
understanding of the molecular principles that govern antibody
specificity remains a major challenge. In this paper, we report the
crystal structure of the cross-reaction complex between the antigen
Guinea fowl lysozyme (GEL) Avian egg
lysozymes have long served as model antigens for investigating the
specificity of immune recognition (for review, see Benjamin et
al.(1984)). Cross-reaction studies using panels of evolutionarily
related avian lysozymes have identified amino acid residues and the
general region of the antigen bound by specific mAbs. Using this test,
the antibodies could be grouped by their binding specificities (Harper et al., 1987; Lavoie et al., 1992). Crystallographic
analysis of several Fab-lysozyme complexes (Amit et al., 1986;
Sheriff et al., 1987; Padlan et al., 1989; Chitarra et al., 1993; Braden et al., 1994) have extended
these characterizations of antigenic determinants (or epitopes), and
common features have emerged from these studies. 1) The
lysozyme-antibody interfaces are large (600-800
Å In the crystal
structure of the cross-reaction complex between Fab F9.13.7 and the
heterologous antigen GEL, we have found unexpectedly that the epitope
bound by F9.13.7 nearly coincides with that bound by the anti-HEL mAb
HyHEL10 (Padlan et al., 1989). However, mAbs F9.13.7 and
HyHEL10 are structurally different in their L and H chains. We discuss
how the two antibodies, with unrelated combining sites, achieve
specific, high affinity binding to nearly the same epitope of lysozyme.
Figure 1:
a, electron
density (2F
Sequencing of the first nine amino acids from the L chain was
carried out by Edman degradation on a 470 Applied Biosystems sequencer.
No sequence data could be obtained for the H chain presumably due to a
blocked amino terminus. Sequences were analyzed using GCG software
(Devereux, 1991). Numbering of the amino acid positions follows the
convention of Kabat et al.(1991).
Molecular
surface areas were calculated using a modified version of the program
MS (Connolly, 1983) with a probe radius of 1.7 Å and standard Van
der Waals' radii for protein atoms. The atomic coordinates of the
HyHEL10
Figure 5:
Overall view of the F9.13.7-GEL (a) and HyHEL10-HEL (b) complexes with the antigens
in similar orientations. Only the variable dimers have been represented
for clarity. The color scheme of the backbone representation is as
follows: lysozyme epitope, purple; L1, brown; L2, lightblue; L3, yellow; H1, darkblue; H2, green; H3, red.
Complex formation induced
only small conformational changes in the antigen polypeptide chain.
Comparison of uncomplexed GEL determined at a 1.9-Å resolution
(Lescar et al., 1994) with bound GEL gives r.m.s. deviations
of 0.80 and 0.86 Å for corresponding main chain atoms, comparable
with the differences observed between the two copies of lysozyme in the
crystal structure of the complex (0.87 Å). The largest deviations
(>1.4 Å) occur for a lysozyme loop (positions 100-104)
at the periphery of the epitope. This loop, however, has no clear
conformation in the structure of uncomplexed GEL (Lescar et
al., 1994), and the observed differences likely reflect its
intrinsic flexibility.
Figure 6:
Schematic view of the intermolecular
contacts (<4 Å) showing the relative disposition of the CDRs
with respect to the epitope for the F9.13.7-GEL (a) and
HyHEL10-HEL (b) complexes. Antigen is represented in gray (a roundedrectangle represents the lysozyme
Figure 2:
Nucleotide and deduced amino acid sequence
of the V
Figure 3:
a,
Figure 8:
Stereo view of the region around Arg-21 in
the F9.13.7-GEL (a) and HyHEL-10-HEL (b)
complexes.
The V The long H3 loop (13 residues), which has the same conformation in
the two noncrystallographically related complex molecules, partially
protrudes into the active site cleft of lysozyme. Three consecutive Tyr
residues encoded by the diversity gene segment are involved in stacking
interactions with aromatic residues from neighboring CDRs (Fig. 1b); the next four residues, at the tip of H3,
form a 3
Figure 4:
Buried surface areas of lysozyme residues
contacting the F9.13.7 and HyHEL10 antibodies upon formation of the
complex. The overall correlation is 61%.
Despite
binding a nearly identical epitope, the two antibodies have no sequence
similarity at any of their CDRs (Fig. 2) and bind the epitope in
different relative orientations (Fig. 5). A topographic
correspondence can be established between the hypervariable loops L3 of
F9.13.7 and H1 of HyHEL10, both of which interact with the same region
of lysozyme (Fig. 6). In addition, the H3 loop of F9.13.7
fulfills an analogous role to that of H2 in HyHEL10, creating a
protrusion that partially fits into the active site cleft of lysozyme (Fig. 1b and 5). On the opposite side of the epitope,
H1 (F9.13.7) substitutes for L3 (HyHEL10), and extensive contacts made
by the H2 region of F9.13.7 are made by L1 and L2 in the
HyHEL10 Several intermolecular hydrogen bonds and
salt bridges are observed in the two antigen-antibody interfaces. In
general, the polar and charged groups are distributed in a similar
fashion at the two antibody combining sites, clustered on a ridge
approximately 10 Å from the geometric center of the
antigen-binding region. Essentially the same epitope residues (which
are identical between GEL and HEL) are involved in polar interactions
with the antibody combining sites. A single salt bridge between
Asp(H32) from the H chain and Lys-97 from lysozyme was found in the
HyHEL10 The overall backbone structures of GEL bound to
F9.13.7 and HEL bound to HyHEL10 are very similar despite the sequence
differences between HEL and GEL (r.m.s. deviation of 0.89 Å for
equivalent C
The reduced
affinity of antibodies F9.13.7 and D11.15 for JQL, which has two amino
acid substitutions at positions 102 and 103 compared with HEL, has led
to the assumption that this region could be bound by the two antibodies
and that their epitopes could overlap. This hypothesis, however, is
inconsistent with the formation of a ternary complex
F9.13.7
Electrostatic model
calculations carried out for the F9.13.7 The side chain of Arg-73 protrudes toward the antibody combining
site in both structures. In the F9.13.7
Figure 7:
Stereo view of the structural environment
around Arg-73 in the F9.13.7-GEL (a) and HyHEL-10-HEL (b) complexes. Lysozyme backbone is shown in white;
antibody backbone is shown in gray (see text for
details).
On the opposite side of the
epitope, Arg-21 binds to a pocket formed by the H2 and L3 loops of
HyHEL10 (Fig. 8b). The The F9.13.7-GEL complex shares common features with other
antibody-antigen complexes whose structures have been reported. The
molecular surfaces brought in contact upon complex formation are of
comparable size, largely complementary in shape, and involve a similar
number of contact residues and hydrogen bonds. The equilibrium
association constant (K Comparative studies of known
antibody structures have led Chothia and Lesk(1987) to propose the
existence of a small number of backbone conformations, or canonical
structures, for five of the six hypervariable loops. The results
presented in this paper further substantiate this hypothesis. Although
the homologous H1 and H2 hypervariable loops of F9.13.7 and D11.15 have
very similar conformations (Fig. 3b), they account for
about half of the corresponding combining site surfaces and are engaged
in interactions with quite different structural environments. This
finding has interesting implications, not only for the modeling of
antibody combining sites (Chothia et al., 1989) but also for
the molecular mechanisms that generate antibody diversity, since a
limited set of main chain conformations for most hypervariable loops
would ensure a potentially unlimited functional repertoire. Further
evidence of heterologous binding is provided by the comparison of the
F9.13.7 Previous studies of
anti-idiotypic antibodies have suggested that in certain cases
functional mimicry could be partially based on a true molecular mimicry
(for review, see Taub and Greene(1992) and Mariuzza and Poljak(1993)),
the most plausible example being provided by a crystallographic study
of anti-angiotensin II antibodies (Garcia et al., 1992). The
data presented here demonstrate that functional mimicry by the two
antibodies is possible even in the absence of any sequence similarity
in their combining sites. Since antigen selection operates on
functional, not structural, conformations, the known difficulties
encountered in evidencing antibody mimics (see, for example, Davis et al.(1992)) suggest that heterologous binding by unrelated
molecular surfaces may be a common phenomenon in antigen-antibody
interactions. This is in agreement with the known degeneracy of immune
responses and germline gene information to provide complementary
structures that bind specifically to an antigenic determinant. Comparison of the F9.13.7- and HyHEL10-bound lysozymes provides an
estimate of the extent to which conformational changes are involved in
producing the observed complementarity of the antigen-antibody
interfaces. The plasticity of the epitope contributes to restore steric
complementarity to differently shaped combining sites. Indeed,
molecular surface adaptability appears to be a general principle
underlying promiscuity in protein-protein interactions (Malby et
al., 1994). In the case of lysozyme, these changes arise primarily
from side chain mobility and are not significantly reflected at the
backbone level. This example and other recent studies of
antigen-antibody interactions (for review, see Davies and Padlan(1992)
and Wilson and Stanfield(1993)) underline the role of conformational
changes in immune recognition and may have important implications for
theoretical approaches to modeling protein-protein interactions. The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s) U20819 [GenBank Link]and U20820[GenBank Link]. The atomic coordinates (code 1FBI) have been deposited in the
Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helix and surrounding loops adjacent to
the lysozyme active site cleft. The epitope of lysozyme bound by
antibody F9.13.7 overlaps almost completely with that bound by antibody
HyHEL10; the same 12 residues of the antigen interact with the two
antibodies. The antibodies, however, have different combining sites
with no sequence homology at any of their complementarity-determining
regions and show a dissimilar pattern of cross-reactivity with
heterologous antigens. Side chain mobility of epitope residues
contributes to confer steric and electrostatic complementarity to
differently shaped combining sites, allowing functional mimicry to
occur. The capacity of two antibodies that have different fine
specificities to bind the same area of the antigen emphasizes the
operational character of the definition of an antigenic determinant.
This example demonstrates that degenerate binding of the same
structural motif does not require the existence of sequence homology or
other chemical similarities between the different binding sites.
(
)and the Fab
fragment from the mAb F9.13.7, raised against HEL.
) and highly complementary. 2) Both the L and H
chains of antibodies make extensive contacts with lysozyme, and the
specificity of binding is determined by the CDRs. 3) The contacting
residues of lysozyme are discontinuous in sequence but form a
contiguous surface. 4) van der Waals' interactions, hydrogen
bonds, and, to a much lesser extent, salt bridges mediate the binding
of antibodies to lysozyme. 5) Small conformational changes in the
antibodies and in lysozyme take place upon complex formation. In
addition, these x-ray studies indicated that antibody cross-reactivity
results from interactions with epitopes that are shared by heterologous
lysozymes or that are closely similar and can be contacted by the
antibody with minor conformational rearrangements.
Crystallization and Data Collection
Preparation
of the proteins and crystallization of the complex have been described
(Lescar et al., 1993). The crystals are monoclinic, space
group P2, unit cell dimensions a = 83.7
Å, b = 195.5 Å c = 50.2
Å,
= 108.5°, with two molecules of the complex
in the asymmetric unit. Crystal instability upon irradiation precluded
merging diffraction data from different crystals. A single data set for
structure refinement was collected at 4 °C using a MARresearch
image plate system and synchrotron radiation from beam line D23 at
LURE, Orsay, France (
= 0.986 Å). A total of 111,931
measurements from 180 images (
= 1°) were reduced
to 30,232 unique intensities (95% complete to 3-Å resolution)
with the MOSFLM package (Leslie, 1990) and the CCP4 suite (SERC, 1986).
The internal merging R-factor is 14% for all data to a nominal
resolution of 3 Å (11% for reflections with F >
(F)).
Structure Determination and Refinement
The crystal
structure was determined at 4-Å resolution by molecular
replacement methods using the program AMoRe (Navaza, 1994) as described
previously (Lescar et al., 1993). The structures of
unliganded GEL (Lescar et al., 1994), the variable dimer of
Fab E225 (Bentley et al., 1990) and the constant dimer of Fab
D1.3 (Fischmann et al., 1991) were used as independent search
models. Real space averaging between the two independent copies of the
complex (SKEW) (Bricogne, 1976) significantly improved the quality of
the density map, allowing the unambiguous tracing of all the CDRs that
had been omitted from the initial models. Refinement was carried out
with XPLOR (Brnger et al., 1987), and
model rebuilding was carried out with the programs FRODO (Jones, 1978)
and QUANTA (Molecular Simulations Inc.). The crystallographic
refinement converged to an R-factor of 19% for 23,703 observed
intensities (F > 2(F)) to a 3-Å
resolution. The present model consists of 1130 amino acid residues and
does not include solvent molecules. The r.m.s. deviations from ideal
bond lengths and angles are, respectively, 0.01 Å and 1.65°.
The electron density of the protein backbone is clearly defined for the
two independent complexes and unambiguous for all the CDRs in contact
with lysozyme (Fig. 1a). The refined coordinates have
been deposited with the Protein Data Bank, Brookhaven, NY (Bernstein et al., 1977), code 1FBI.
- F
) map contoured at 1
of the H3
hypervariable loop of F9.13.7. b, stereo view of the contacts
made by H3 (solidlines) with lysozyme (dashedlines). Tyrosine residues of neighboring hypervariable
loops making stacking interactions are also shown (see
text).
Nucleotide and Protein Sequences of F9.13.7
Total
mRNA was purified from 2 10
hybridoma cells by the
method of acid guanidium thiocyanate extraction (Chomczynski and
Sacchi, 1987). cDNA was produced from 5 mg of total RNA using
Superscript Reverse Transcriptase (Life Technolgies, Inc.) and 25 pmol
of immunoglobulin H or L chain constant region specific primers (Huse
and Lerner, 1989). The cDNA was amplified by polymerase chain reaction
using the same 3` primer and a combination of eight 5` primers
described by Huse and Lerner(1989), and the amplified products were
cloned into polymerase chain reaction II plasmids (Invitrogen).
Nucleotide sequencing was performed by the dideoxy chain termination
method (Sequenase Sequencing kit, U. S. Biochemical Corp.). Sequencing
was performed in both directions and confirmed by comparison of at
least two clones obtained from separate polymerase chain reactions. The
nucleotide sequences of the variable regions of the H and L chains have
been deposited with GenBank (accession numbers U20819 and U20820).
Modeling
Electrostatic energy calculations were
carried out as described by Pellegrini and Doniach(1993). A native-like
conformation (within 3 Å, r.m.s.) was found to have the lowest
electrostatic energy among several thousand conformations sampled
during the docking search for the F9.13.7GEL and HyHEL10
HEL
complexes. The models of the chimeric low affinity complexes
HyHEL10
JQL and F9.13.7
PHL were built by superimposing the
crystallographic coordinates of the unliganded lysozyme (JQL, Houdusse
(1992); PHL, Lescar et al.(1994)) on the crystal structures of
the complex. In all cases, a short relaxation stage (to avoid
significant departure from experimental coordinates) was followed by a
molecular dynamics simulation carried out at 300 K for 100 fs,
extracting the binding energy per residue every 10 fs.
HEL complex were taken from the Protein Data Bank, code
3HFM.
The Overall Structure
The quaternary structure
of the F9.13.7GEL complex resembles that of other lysozyme-Fab
complexes. mAb F9.13.7 binds lysozyme through a large contact surface
formed by the CDRs at the amino-terminal end of the variable domain
(see Fig. 5a). The two independent copies of the
complex display systematic variations in overall temperature factors
arising from a different packing environment (Lescar et al.,
1993). Their backbone structures, however, are closely similar. The
overall r.m.s. deviation between all 552 equivalent C
coordinates
is 0.99 Å (0.62 Å excluding the constant (C
1
+ C
) region). The two Fabs present the same extended
conformation with an elbow bend between the variable and constant
domains of 175° in both complexes.
The Lysozyme-Fab Interface
Antigen-antibody
contacts are extensive and involve many residues. The epitope is
discontinuous, consisting of 13 exposed residues from a central
-helix (positions 88-99) and three surrounding loops
(residues 15-21, 73-77, and 100-103). The area of
lysozyme that is in contact with the antibody is 760
Å
. Twelve residues from the H chain and four from the
L chain contribute to the binding of lysozyme (see Fig. 5a). This asymmetry is reflected by the buried
surface areas upon complex formation (570 Å
for the H
chain and 200 Å
for the L chain). Indeed, only two
lysozyme residues (Arg-73, Asn-77) interact with the L1 and L3
hypervariable loops, whereas all other lysozyme residues contact
residues from the H chain (see Fig. 6a). A large number
of electrostatic interactions are observed at the interface. Three salt
bridges and 12 hydrogen bonds are formed between antibody and lysozyme
residues (Table 1). The majority of these polar contacts involve
antibody residues from the H2 and H3 hypervariable regions.
-helix) and antibody in color. Intermolecular salt linkages are
indicated with thickarrows.
Somatic Mutations
The antibody F9.13.7 expresses a
member of the V124 variable, DFL16.1 diversity, and
J
4 joining gene segment families in the H chain and
V
10-Ars-A and J
2 in the L chain. Comparison with the germline
sequences (Fig. 2) suggests that somatic mutations make a
limited contribution to binding affinity. Five nucleotide differences
are observed in each of the two variable segments. Four of these (at
positions L23, L68, H50, and H63) are silent mutations, and three
others (L38, L76, and H40) involve framework residues that do not
contact the antigen. Two expressed mutations occurring in the H2 loop,
Thr(H58)
Pro and Gln(H62)
Glu, affect positions that are
not in contact with lysozyme. Furthermore, these substitutions do not
affect the conformation of the loop compared with that of D11.15,
another anti-lysozyme antibody that has the germline-encoded amino
acids at these positions (Fig. 3). Indeed, the only change of
somatic origin affecting a residue contacting lysozyme involves L chain
Tyr(L92) (Asn in the V
10-Ars-A sequence), partially stacked
against the
-guanido group of Arg-73 (see Fig. 8a).
(a) and V
(b) domains of
F9.13.7. The nucleotide sequences are compared with those of the
germline segments V
10-Ars-A and V
124. Germline-encoded
amino acids that differ from those of F9.13.7 are indicated above the nucleotide sequence. The protein sequence of the V
domain
of F9.13.7 is compared with those of anti-arsonate antibody R19.9 and
HyHEL10. The protein sequence of the V
domain of F9.13.7 is
compared with those of anti-lysozyme antibodies D11.15 and HyHEL10. Dots indicate nucleotide identities; dashes indicate
amino acid identities. Numbering is as in Kabat et al.(1991).
The nucleotides corresponding to the first six amino acids of the
V
domain of F9.13.7 have not been
sequenced.
-carbon trace of the
variable regions of D11.15 (left) bound to PHL (in red) and of F9.13.7 (right) bound to GEL (in yellow). The hypervariable loops H1 and H2 of D11.15 (in green) and of F9.13.7 (in purple) contact different
regions of the lysozyme surface. Side chain atoms are shown for the
lysozyme loop at positions 100-103. b, main chain atoms
of the superimposed H1 and H2 loops are color-coded as
above.
Conformation of the Complementarity-determining
Regions
The L chain of F9.13.7 is very similar to that of
CRI-positive anti-p-azobenzenearsonate antibodies
(Sanz and Capra, 1987). The amino acid sequences of V
F9.13.7 and the anti-arsonate R19.9 (Lascombe et al.,
1992) differ at only five positions (Fig. 2), and the
corresponding C
coordinates can be superimposed with a r.m.s.
deviation of 0.58 Å. In particular, the three hypervariable loops
have the same conformation with only two amino acid substitutions in
the L3 loop, reinforcing the concept that their H chain partners are
the primary determinants of specificity (Siekevitz et al.,
1983).
domain of F9.13.7 resembles that of
anti-HEL antibody D11.15 (Chitarra et al., 1993). The H1 and
H2 loops of these two antibodies are closely similar, although they
contribute significantly (50% of the corresponding contact surfaces) to
binding nonoverlapping regions of lysozyme (Fig. 3). This
comparison illustrates how the same structural elements can confer
binding specificity in quite different stereochemical environments.
This lends further support to the canonical loop hypothesis (Chothia et al., 1989), according to which a limited set of
hypervariable loop conformations is sufficient to generate diversity.
-helical turn that makes contact with lysozyme
residues (in particular with Trp-63 on the enzyme active site), and the
subsequent three amino acids adopt an extended conformation that
connects back with the framework
-strand.
Comparison of F9.13.7-GEL and HyHEL10-HEL
Complexes
The epitope of GEL defined by F9.13.7 extensively
overlaps with that of the antibody HyHEL10 (Padlan et al.,
1989). The same 12 residues of the antigen (see Fig. 6) make 14
of the 15 intermolecular hydrogen bonds or salt links observed in each
complex (Table 1). The surface areas buried upon Fab-lysozyme
interaction are about the same for the two complexes (770 Å for the antibody and 760 Å
for lysozyme). The
overlap between the lysozyme epitopes bound by mAbs F9.13.7 and HyHEL10
can be numerically evaluated by plotting the surface lost by each
lysozyme residue upon complex formation (Fig. 4).
HEL complex. The unusually short H3 loop of HyHEL10
contributes a single residue (Trp(H95)) to binding, as does the L1 loop
of F9.13.7 (Tyr(L32)).
HEL complex, despite the presence of several exposed
charged residues (Padlan et al., 1989). The situation is
different for F9.13.7, since the H2 loop contains two germline-encoded
Asp and one Glu that form ion pairs with basic residues of the epitope (Fig. 6a). Thus, Asp(H52) interacts with Lys-96,
Asp(H54) with His-15, and Glu(H50) with Lys-97. The (Asp(H54)-His-15)
salt bridge, however, is exposed to the solvent and could be weakened
by hydration.
positions), indicating that heterologous binding can
be achieved with little conformational change in the lysozyme backbone.
Complex formation, however, entails more significant differences in
side chain conformations. The equivalent C
atoms of the 12
lysozyme residues common to both epitopes can be superimposed with
r.m.s. deviations of 0.8-0.9 Å, but these values rise to
1.5-1.6 Å when main chain and side chain atoms are included
in the comparisons. The flexibility of side chains permits surface
variations on lysozyme that match steric differences between the two
antibody combining sites and allows for complementarity of the
antigen-antibody interfaces.
Fine Specificity of F9.13.7
The association
constant of F9.13.7 for HEL, as determined by microcalorimetric
techniques, is 3.4 10
M
(Tello et al., 1993). Cross-reactions of F9.13.7 with
other avian lysozymes indicated that the relative affinity for GEL,
bobwhite quail, and California quail lysozymes were similar to that for
HEL, whereas much weaker or no reactions were observed for JQL, PHL,
and turkey lysozymes (Tello et al., 1990).
HEL
D11.15, observed by gel filtration chromatography
(data not shown). In the crystal structures of the Fab D11.15
PHL
(Chitarra et al., 1993) and the Fab F9.13.7
GEL
complexes, residues from the lysozyme loop 100-103 interact with
the two antibody combining sites, in agreement with the immunochemical
mapping results. Nevertheless, simultaneous binding by the two
antibodies is possible (Fig. 3) with only a minor conformational
adjustment of the loop backbone needed to avoid steric clash.
Cross-reactivity Pattern of F9.13.7 and
HyHEL10
Despite binding a common set of residues, the F9.13.7
and HyHEL10 antibodies display a different pattern of fine specificity
toward heterologous lysozymes. Both antibodies bind HEL and GEL with
comparable affinities, but react differently with JQL and PHL. The
F9.13.7JQL and HyHEL10
PHL complexes have similar high
affinities, whereas the association constant of the F9.13.7
PHL
and HyHEL10
JQL complexes is significantly weaker (Smith-Gill et al., 1984; Tello et al., 1990). Thus, epitope
mapping based on binding assays would point to different antigenic
determinants (functional epitopes) on lysozyme, although the two
antibodies recognize a very similar structural region, illustrating how
different analytical approaches may lead to different perceptions of a
protein epitope (Van Regenmortel, 1989).
GEL and HyHEL10
HEL
complexes suggest that a few lysozyme residues, different for each
antibody, contribute the most to binding. In the HyHEL10
HEL
complex, three residues of the epitope (Arg-21, Lys-97, and Asp-101)
account for half of the total average binding energy, in agreement with
mutagenesis studies (Kam-Morgan et al., 1993), showing that
substitutions of Arg-21 or Asp-101 significantly decrease the binding
constant for HyHEL10. On the other hand, five residues (His-15, Arg-73,
Lys-96, Lys-97, and Asp-101) contribute 80% of the F9.13.7
GEL
binding energy. Leaving aside the expected contribution of amino acids
involved in intermolecular salt bridges (His-15, Lys-96, and Lys-97),
these results suggest that the arginine residues at positions 21 and 73
are important in accounting for the difference in antibody specificity.
GEL complex, it is mostly
occluded from the solvent by stacking interactions with Tyr(L92),
which, together with Tyr(L32) and Leu(L94), form a ridge on the
antibody surface (Fig. 7a). In contrast, the same
arginine is largely exposed to the solvent in the HyHEL10
HEL
interface (Fig. 7b). Furthermore, the planar ring of
active site residue Trp-62 is oriented differently in the two bound
lysozymes: perpendicular to the interface in the F9.13.7-GEL complex
(where it is stacked against the Arg-73 side chain) and roughly
parallel to the interface (and to the aromatic ring of Tyr(H53)) in the
HyHEL10
HEL complex. The structural environment of Arg-73 is
therefore consistent with the different binding constants of the
antibodies for PHL and turkey lysozyme, for which two amino acid
changes occur at the epitope with respect to HEL (His-15
Met/Leu) and (Arg-73
Lys).
-guanido group of the
arginine side chain is within hydrogen bonding distance of Tyr(H50),
whereas its aliphatic moiety is partially screened from the solvent by
the planar rings of Trp(L94) and Tyr(H58). The F9.13.7 combining site
lacks a similar binding pocket; the Arg-21 side chain is in contact
with the aromatic ring of Tyr(H32) but otherwise exposed to the solvent (Fig. 8a). The structural context around Arg-21 could
account for the weak reaction of HyHEL10 with JQL, which has a glutamic
acid at this position (Kam-Morgan et al., 1993). The same
substitution would instead have a limited effect on F9.13.7 reactivity
because of a stereochemically permissive environment. Two additional
amino acid differences between HEL and JQL at positions close to the
antigen-antibody interface, Gly-102 (HEL)
His (JQL) and Asn-103
(HEL)
His (JQL), appear to be less critical for binding, since
replacements of Gly-102 have no effect on the affinity of HEL for
HyHEL10 (Kam-Morgan et al., 1993).
= 10
M
) is also typical of previously
studied antigen-antibody complexes (K
= 10
-10
M
).
GEL and HyHEL10
HEL complexes. 12 out of 13 lysozyme
residues in contact with the F9.13.7 combining site also interact with
that of HyHEL10, despite the absence of amino acid sequence homology in
the CDRs of the two antibodies. Other examples of unrelated proteins
binding a same set of amino acid residues have been reported. Bentley et al.(1990) found that seven amino acid residues of anti-HEL
antibody D1.3 are involved in interactions with both the antigen and an
anti-idiotypic antibody, whereas the structure of the complex between
the monomeric human growth hormone and its dimeric receptor (de Vos et al., 1992) showed that the same set of residues on the two
receptor monomers binds a different structure on the hormone. More
recently, Malby et al.(1994) found that approximately 80% of
the binding site of NC10 antibody on neuraminidase overlaps with that
of the NC41 antibody and Braden et al.(1994) showed that mAbs
D44.1 and HyHEL-5 bind a nearly identical epitope on lysozyme (in this
case the H chains of the two mAbs result from the expression of the
same V
gene although their H3 regions and L chains are
unrelated, giving different combining sites and interactions with
lysozyme). In none of these examples do cross-reacting molecular
surfaces display obvious structural similarity.
We thank J. D'Alayer for protein sequencing and
our collegues G. A. Bentley and F. A. Saul for help in data collection
and stimulating discussions. We also thank the LURE staff for access to
synchrotron beamline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.