This information is current as
of June 17, 2017.
Structural Analysis of Der p 1−Antibody
Complexes and Comparison with Complexes
of Proteins or Peptides with Monoclonal
Antibodies
Tomasz Osinski, Anna Pomés, Karolina A. Majorek, Jill
Glesner, Lesa R. Offermann, Lisa D. Vailes, Martin D.
Chapman, Wladek Minor and Maksymilian Chruszcz
Supplementary
Material
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2015; 195:307-316; Prepublished online 29 May
2015;
doi: 10.4049/jimmunol.1402199
http://www.jimmunol.org/content/195/1/307
The Journal of Immunology
Structural Analysis of Der p 1–Antibody Complexes and
Comparison with Complexes of Proteins or Peptides with
Monoclonal Antibodies
Tomasz Osinski,*,† Anna Pomés,‡ Karolina A. Majorek,*,† Jill Glesner,‡ Lesa R. Offermann,x
Lisa D. Vailes,‡ Martin D. Chapman,‡ Wladek Minor,* and Maksymilian Chruszczx
H
ouse dust mites are a common source of indoor allergens
and a major cause of perennial asthma worldwide (1, 2).
House dust mites are common in households around
the world and can be found in beds, carpets, and soft furniture.
Members of the Dermatophagoides genus feed on dander and
small particles of shed skin, which is commonly present in
households. Some of their digestive enzymes are potent proteases
*University of Virginia, Charlottesville, VA 22908; †Adam Mickiewicz University,
61-712 Poznan, Poland; ‡Indoor Biotechnologies, Inc., Charlottesville, VA 22903;
and xUniversity of South Carolina, Columbia, SC 29208
ORCID: 0000-0001-7521-5485 (M.C.).
Received for publication August 26, 2014. Accepted for publication April 20, 2015.
This work was supported by National Institute of Allergy and Infectious Diseases/
National Institutes of Health Grant R01 AI077653 (to A.P., M.D.C., and M.C.) and in
part by National Institutes of Health Grant GM53163. The content is solely the
responsibility of the authors and does not necessarily represent the official views
of the National Institutes of Health. The structural results in this study are derived
from work performed at Argonne National Laboratory, at the Structural Biology
Center of the Advanced Photon Source. Argonne National Laboratory is operated
by University of Chicago Argonne, LLC, for the U.S. Department of Energy, Office
of Biological and Environmental Research under Contract DE-AC02-06CH11357.
Use of the Advanced Photon Source, an Office of Science User Facility operated for
the U.S. Department of Energy Office of Science by Argonne National Laboratory,
was supported by U.S. Department of Energy Contract DE-AC02-06CH11357. Use
of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and by Michigan Technology Tri-Corridor Grant 085P1000817.
The atomic coordinates and structure factors of structures presented in this article
have been submitted to the Protein Data Bank (http://www.rcsb.org) under accession
numbers 4PP1, 4PP2, and 4POZ.
Address correspondence and reprint requests to Dr. Maksymilian Chruszcz, University of South Carolina, 631 Sumter Street, Columbia, SC 29208. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: LV, low viscosity; NCBI, National Center for
Biotechnology Information; PDB, Protein Data Bank; PEG, polyethylene glycol;
PISA, Proteins, Interfaces, Structures and Assemblies; VH, H chain of V region of
Ig; VL, L chain of V region of Ig.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402199
that are abundant in the feces of dust mites and are highly allergenic. Der p 1 is a cysteine protease and a major allergen (3).
Chronic exposure to Der p 1 occurs by inhalation and may lead to
the production of IgE Abs in susceptible atopic individuals. Der p
1 catalyzes the cleavage of the amide linkages in substrates such
as a1-antitrypsin, the CD23 receptor on human B cells, the IL-2
receptor (CD25) on human T cells, and the Der p 1 propolypeptide
sequence (4). Strong evidence suggests that Der p 1–related
cleavage of these receptors contributes to its allergenicity (5, 6).
Structures of recombinant Der p 1 in both the proenzyme and
mature forms were previously determined (7–9). The structure of
natural Der f 1, which shares 81% sequence identity to Der p 1, was
also determined (9). Additionally, structures of natural Der f 1 and
natural Der p 1 in complex with the Fab fragment of the crossreactive mAb 4C1 were also elucidated (10). In this study, we present the crystal structures of Der p 1, isolated from its natural source,
complexed with the Fab fragment of 5H8 (Der p 1–5H8), Der p 1
complexed with the Fab fragment of 10B9 (Der p 1–10B9), and the
Fab fragment of mAb 10B9 alone. Both 10B9 and 5H8 are species
specific, whereas the 4C1 Ab is cross-reactive between Der p 1 from
Dermatophagoides pteronyssinus and Der f 1 from Dermatophagoides
farinae. This enabled the Der p 1 epitopes for mAbs 10B9, 5H8, and
4C1 to be compared with the corresponding surface on Der f 1 (9, 10).
It was discovered that the Der p 1 epitopes, which bind 4C1 and 10B9
Abs, overlap and these two Abs compete for the same binding site
(11). The 5H8 Ab, however, binds to the epitope located on a different
side of Der p 1 and does not compete with 4C1 or 10B9 for binding
(11). The binding interfaces of Der p 1 with mAbs 4C1, 5H8, and
10B9 with the binding interfaces of all currently known structures of
complexes of proteins or peptides with mAbs were also compared.
Materials and Methods
Production and purification of proteins
Der p 1 was purified from D. pteronyssinus mite culture as described
previously for Der f 1 (9, 10). Briefly, Der p 1 was purified from spent
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Der p 1 is a major allergen from the house dust mite, Dermatophagoides pteronyssinus, that belongs to the papain-like cysteine
protease family. To investigate the antigenic determinants of Der p 1, we determined two crystal structures of Der p 1 in complex
with the Fab fragments of mAbs 5H8 or 10B9. Epitopes for these two Der p 1–specific Abs are located in different, nonoverlapping
parts of the Der p 1 molecule. Nevertheless, surface area and identity of the amino acid residues involved in hydrogen bonds
between allergen and Ab are similar. The epitope for mAb 10B9 only showed a partial overlap with the previously reported
epitope for mAb 4C1, a cross-reactive mAb that binds Der p 1 and its homolog Der f 1 from Dermatophagoides farinae. Upon
binding to Der p 1, the Fab fragment of mAb 10B9 was found to form a very rare a helix in its third CDR of the H chain. To
provide an overview of the surface properties of the interfaces formed by the complexes of Der p 1–10B9 and Der p 1–5H8, along
with the complexes of 4C1 with Der p 1 and Der f 1, a broad analysis of the surfaces and hydrogen bonds of all complexes of Fab–
protein or Fab–peptide was performed. This work provides detailed insight into the cross-reactive and specific allergen–Ab
interactions in group 1 mite allergens. The surface data of Fab–protein and Fab–peptide interfaces can be used in the design
of conformational epitopes with reduced Ab binding for immunotherapy. The Journal of Immunology, 2015, 195: 307–316.
308
D. pteronyssinus mite culture extract (∼ 100 g/1 PBS) using affinity
chromatography through a 4C1 mAb column. The mAbs 5H8 and 10B9
were fragmented commercially by GenicBio (Shanghai, China) and Strategic BioSolutions (Newark, DE), respectively. The fragmentation was
performed using papain, and the resulting Fabs were purified by protein A
affinity chromatography. The Fab from mAb 5H8 was further purified by
gel filtration (Sephadex G-75). Both Der p 1–5H8 and Der p 1–10B9
complexes were prepared using the same protocol. In each case, the allergen was mixed with the Fab fragment of Ab in a 1:1 molar ratio and
incubated at 4˚C for 16 h for Der p 1–10B9 and 30 min for Der p 1–5H8.
After incubation, the solution was concentrated using an Amicon Ultra
concentrator (Millipore) with a 10,000-Da molecular mass cutoff and
purified on a Superdex 200 column attached to an ÄKTA FPLC system
(GE Healthcare). A solution composed of 10 mM Tris-HCl and 150 mM
NaCl at pH 7.5 was used for gel filtration of both complexes. After gel
filtration, fractions containing Der p 1–5H8 and Der p 1–10B9 were
concentrated to ∼5 mg/ml. The 10B9 Fab fragment, used for crystallization of the Ab fragment alone, was also purified on a Superdex 200 using
10 mM Tris-HCl, 50 mM NaCl (pH 7.5). Prior to crystallization, the 10B9
Fab fragment was concentrated to 8 mg/ml.
Crystallization
Data collection, structure determination, refinement, and
validation
Data were collected at the Structural Biology Center Collaborative Access
Team 19-BM and 19-ID beamlines (12), as well as at the 21-ID-D beamline
of the Life Sciences Collaborative Access Team at the Advanced Photon
Source, Argonne National Laboratory. All structures were determined by
molecular replacement using HKL-3000 (13), which incorporates MOLREP (14) and some of the programs included in the CCP4 package (15).
The structure of the Fab fragment of mAb 10B9 was determined using the
Fab fragment of mAb 4C1 (Protein Data Bank [PDB] code 3RVT) as
a starting model. Structures of Der p 1 in complex with the Fab fragments
of mAb 5H8 and 10B9 were determined utilizing the Der p 1 allergen
complexed with the Fab fragment of mAb 4C1 (PDB code 3RVW) (10) as
the start model. The sequences of the mAbs were obtained by sequencing
reverse-transcribed mRNA isolated from hybridomas producing the mAb
(9). The models were refined with COOT (16) and REFMAC5 (17). TLS
groups used in the refinement of Der p 1 with the Fab fragment of mAb
10B9 were generated using the TLSMD Web server (18). All three
structures were validated with MOLPROBITY (19) and ADIT (20).
Previous results showed that natural Der p 1 binds a calcium ion (10), and
the metal bound by Der p 1–10B9 and Der p 1–5H8 complexes was also
determined as calcium. Data collection and refinement statistics can be
found in Table I. The atomic coordinates and structure factors for all the
structures reported in the present study were deposited in the Protein Data
Bank (www.rcsb.org) with the following accession codes: 4PP1 (Der p 1–
5H8), 4PP2 (Der p 1–10B9), and 4POZ (10B9).
Structure analysis of Der p 1 with 10B9 and Der p 1 with 5H8
Sequences of the protein structures that are similar to the H and L chains of
the Fab fragments of mAb 10B9 and 5H8 were obtained by a Basic Local
Alignment Search Tool (BLAST) (21–23) search with an expectation value
of 1e210 against the pdbaa BLAST database (24). Structures of the most
similar proteins were downloaded, and STRIDE (25) was used to determine their secondary structures. The Abs containing an a helix in the CDR
H3 were superposed in PYMOL (26) and their CDRs were analyzed. The
amino acids involved in the formation of hydrogen bonds between proteins
were identified with Proteins, Interfaces, Structures and Assemblies (PISA)
(27), and a cutoff distance of 3.3 Å for hydrogen bonds was chosen. The
salt bridges between Der p 1 and the Fabs of 10B9 and 5H8 were identified
by PISA (Gibbs free energy based) and VMD (distance based) (28). A salt
bridge was considered to be formed when the distance between any of the
oxygen atoms of acidic residues (carboxylate ions; acceptors) and the
nitrogen atoms of basic residues (ammonium or guanidinium ions; donors)
were within the cut-off distance of 4.0 Å (29–31).
Structures of Fab fragments of mAbs complexed with various macromolecules were obtained by running a PSI–BLAST search against the
pdbaa database using the sequences of 5H8 and 10B9. Redundancy was
removed and two datasets were created: the first, which contains structures
with resolution of 3.0 Å or better, and the second, which contains structures with resolution of 3.5 Å or better. Preliminary analysis of the epitope
and paratope content and hydrogen bonds between them were virtually
identical for both datasets. All of the analyses were performed simultaneously on both datasets; however, the results from the first dataset, which
contains 314 structures of Fab fragments in complex with protein or
peptides solved at 3.0 Å resolution or better, were chosen for this study and
referred to hereafter as the dataset. PISA was used to estimate the interfaces and hydrogen bonds formed by structures in the dataset. Amino acids
were considered to be part of the interface when the calculated buried
surface area for a particular amino acid was .10 Å2 or was involved in
forming hydrogen bonds.
The VMD program was used for structural conservation analysis between
Der p 1 and Der f 1. Der p 1 (3F5V), Der f 1 (3D6S), Der f 1–4C1 (3RVV),
Der p 1–4C1 (3RVW), Der p 1–10B9 (4PP2), and Der p 1–5H8 (4PP1)
were the structures used for comparison.
RING (32) was used as a tool for analyzing residue interaction networks
to describe the protein’s three-dimensional structure and the nature of interactions (e.g., hydrogen bonds, van der Waals contacts, p–cation interactions
and p–p stacking interactions).
Other techniques
Residues on the protein’s surface have been identified with PYMOL. The
pairwise protein sequence identity and similarity were calculated using the
EMBOSS (33) package.
The frequencies of amino acids are shown per 100 surface-exposed
amino acids found in all protein chains of Fab–protein or Fab–peptide
complexes reported in the PDB database (as of 2013). Amino acid frequencies calculated this way were used to estimate the expected amino
acid content of the epitopes and paratopes to compare it with the observed
amino acid content of the interfaces.
A program was written in python (34) with the use of numpy (35),
matplotlib (36), and mmlib (37) libraries to generate three types of plots:
1) bar plots for comparison between observed and expected distribution of
the amino acids among epitopes and paratopes, 2) rainbow-colored bar
plots showing the relative amount of surface area contributed to the Ag–Ab
interface by each type of amino acid, and 3) array plots for displaying the
number of hydrogen bonds between epitopes and paratopes as a grayscale.
The values of the expected number of amino acids in the interfaces for the
bar plots were derived from the frequencies of amino acid occurrences in
all protein sequences found in BLAST nr database. The dataset obtained in
the first step of the structural analysis was used for plots 2 and 3. The area
contributed by a particular amino acid to the interface in the rainbowcolored plot was calculated with PISA. The number of hydrogen bonds
in Ag–Ab complexes shown in plot 3 was also calculated with PISA. Some
of the code was developed and used in a previous work (10).
Molecular Operating Environment (38) was used to verify the potential
hydrogen bonds involved in creating the interfaces between Der p 1 and
both Fab fragments of mAb 5H8 and 10B9.
Results
Der p 1 with the Fab fragment of mAb 5H8 and Der p 1 with the
Fab fragment of mAb 10B9 were purified, crystallized, and their
structures were determined (Table I). Space groups were identified
as P21212 for the Fab fragment of mAb 10B9 and P21 for the Der
p 1–5H8 and Der p 1–10B9 complexes. Previously, we were able
to determine the crystal structure of Der p 1 complexed with the
Fab fragment of mAb 4C1 (10). Fig. 1A shows the relative position
of the Fab fragments of 4C1, 10B9, and 5H8 Abs in complex with
Der p 1. More than 70% of the 10B9 binding epitope overlaps with
the epitope that binds the 4C1 Ab; the epitope binding 5H8 Ab is
located on a different part of Der p 1.
The electron density found in close proximity to Asn52 in each of
the Der p 1 chains was interpreted as N-acetylglucosamine in the
structures of both complexes. Additionally, it was discovered that
the same, or similar, residue binds N-acetylglucosamine in the
structures of group 1 dust mite allergens previously solved, where
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Crystallization of Der p 1–10B9, Der p 1–5H8, and 10B9 was performed at
293 K. Crystals were grown using the hanging drop vapor diffusion
method. The crystallization drops were a 1:1 mixture of the protein solution and the precipitant solution from the wells (100 mM MES, 10% [w/v]
polyethylene glycol [PEG] 6000, 5% MPD at pH 7.0 for the Der p 1–5H8
complex, 100 mM sodium acetate, 8% [w/v] PEG 4000, 15% MPD at pH
4.5 for the Der p 1–10B9 complex, and in the case of 10B9, 100 mM
sodium citrate, 15% [w/v] PEG 6000 at pH 5.5 was used). Prior to data
collection, Der p 1–5H8, Der p 1–10B9, and 10B9 crystals were cryoprotected in LV CryoOil solution (MiTeGen, Ithaca, NY), a 1:1 mixture
of Paratone-N and mineral oil, or well solution, respectively, then immediately cooled in liquid nitrogen.
ANTIGENIC STRUCTURE OF Der p 1
The Journal of Immunology
309
Table I. Data collection and refinement statistics
Structure (PDB Code)
Der p 1-10B9 (4PP2)
Der p 1-5H8 (4PP1)
P21212
67.0, 121.8, 55.0
90.0, 90.0, 90.0
1.75 (1.75–1.78)
0.069 (0.633)
27.0 (2.2)
99.0 (96.9)
5.4 (4.7)
P21
50.7, 74.9, 184.1
90.0, 97.4, 90.0
2.74 (2.74–2.79)
0.139 (0.684)
14.5 (2.3)
99.2 (97.9)
4.3 (4.2)
P21
47.7, 73.3, 200.3
90.0, 91.1, 90.0
3.00 (3.00–3.05)
0.165 (0.653)
10.0 (2.3)
99.9 (100.0)
4.1 (4.2)
1.75
43428
17.2/20.7
2.74
34176
20.0/26.1
3.00
26581
21.9/26.5
3364
9866
30
22
9748
48
74
32
47
30
34
50
78
27
0.018
1.8
0.007
1.2
0.009
1.2
449
25
Numbers in parentheses refer to the highest resolution shell.
rmsd, root mean square deviation.
in the case of Der f 1 N-acetylglucosamine was bound by Asn53
(9, 10). An exception was found in a structure where Asn52 was
replaced by Gln52 (8). The N-acetylglucosamine binding site is far
from the 4C1, 10B9, or 5H8 epitopes and does not interfere with
the binding of these Abs.
Three well-ordered CDRs per Ab chain are involved in creating
the interface areas (Supplemental Fig. 2). According to a relatively
recent (2011) classification (39), the conformations of the CDRs
in the Fab fragments of mAb 10B9 and 5H8 can be categorized
into the same categories as 4C1 (10): CDR L1, L1-11-2; CDR L2,
L2-8-1; CDR L3, L3-9-cis7-1; CDR H1, H1-14-1; CDR H2, H2-9-1.
Comparison of epitope–paratope interactions
Der p 1–5H8. Crystals of Fab 5H8 alone were not obtained. In the
structure of the Der p 1–5H8 complex, the epitope binding 5H8 to
Der p 1 is located far from the Der p 1’s active site. The buried
surface area at the interface of Der p 1 and 5H8 is ∼910 Å2. The
interface areas contributed by the H and L chains are ∼75 and
25%, respectively (Table II). Der p 1 epitope binding 5H8 Ab is
formed by 19 aa, where 16 residues from Der p 1 interact with 15
residues from the H chain and 6 residues from Der p 1 interact
with 7 residues from the L chain of 5H8 Ab. Amino acid composition of the epitope on the surface of Der p 1 (Fig. 1B–G)
where the Fab of mAb 5H8 is bound differs significantly from
those epitopes where the Fab of mAb 4C1 and 10B9 are bound.
Fifteen hydrogen bonds were identified between Der p 1 and 5H8
Ab. Residue Arg51 of Der p 1 creates hydrogen bonds with both
the L chain of V region of Ig (VL) and H chain of V region of Ig
(VH) of mAb 5H8 (Gly91 and Asp103, respectively). Some of the
residues, such as Ser114 with Tyr32 and Tyr50 and C-terminal
Leu222 with Lys92, form hydrogen bonds only with the VL,
whereas residues Gln53, Ser54, Gln84, Tyr93, Ala108, Gln109, Arg110,
Gly112, and Ile113 form hydrogen bonds only with the VH of 5H8
Ab (Table II). Supplemental Figs. 3 and 4 demonstrate that hydrogen bond formation is quite specific, where a tyrosine residue
from the Ab tends to form hydrogen bonds with an arginine residue from the Ag. This occurrence can be observed at the inter-
faces between Der p 1–4C1 and Der p 1–5H8, but not in Der p 1–
10B9 complex (see Fig. 3 and our previous work in Ref. 10).
Arg51 forms cation–p interactions with Tyr32 of VL and Tyr102 of
VH. The amino acids that make up mAb 5H8’s paratope of the
5H8–Der p 1 interface are tyrosine, arginine, aspartate, glutamine,
lysine, and a relatively high content of glycine. Phe111 of Der p 1
forms p–p stacking interactions with Trp52 (edge-to-face orientation) and Tyr 102 (face-to-edge orientation) of V H , as well as
Tyr47 of Der p 1. The Fab fragment of mAb 5H8, which is similar
to the Fab fragment of mAb 10B9, is also a species-specific Ab. It
binds to a different fragment of Der p 1, and the epitope for this
Ab does not overlap with the epitopes for the 10B9 and 4C1 Abs.
Der p 1–10B9. The epitope for the Fab fragment of mAb 10B9 is
also located far from the catalytic site of the enzyme (Cys34). The
buried surface area at the interface of Der p 1 and 10B9 is 815 Å2,
and the interface areas contributed by the H chain and L chain are
∼75% for the former and 25% for the latter (Table III). The Der p
1 epitope that binds 10B9 is formed by 17 amino acids, where 15
residues from Der p 1 interact with 11 residues from the H chain
and 5 residues from Der p 1 interact with 5 residues from the L
chain of 10B9.
The uncomplexed Fab fragment of mAb 10B9 crystallized in
the orthorhombic space group P21212. The overall conformation is
almost identical to the Fab fragment of mAb 10B9 that crystallized in complex with Der p 1, but there are conformational differences between the CDRs. A superposition of CL, CH, VL, VH
from the 10B9 crystal and corresponding chains from 10B9 from
the complex with Der p 1 have a root mean square deviation of
0.8 Å. CDRs L1 (residues 23–34), L2 (residues 49–56), L3 (residues
88–97), H1 (residues 23–36), and H2 (residues 50–67) have the
same side chain conformations and almost identical placement
of Ca atoms in both the nonbonded and bound Ab structures.
However, CDR H3 (residues 98–110) has a very rare conformation
in the binding area, an a helix. A comparison of uncomplexed
10B9 with Der p 1–10B9 complex revealed that the CDR H3
undergoes a few significant conformational changes upon binding
to Der p 1. The change is especially well visible for residues
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Data collection
Space group
Cell dimensions: a, b, c (Å)
a, b, g (˚)
Resolution (Å)
Rsym
I/sI
Completeness (%)
Redundancy
Refinement
Resolution (Å)
No. reflections
Rwork/Rfree (%)
No. atoms
Protein
Ligand/ion
Water
B factors (Å2)
Protein
Ligand/ion
Water
rmsd
Bond lengths (Å)
Bond angles (˚)
10B9 (4POZ)
310
ANTIGENIC STRUCTURE OF Der p 1
hydrogen bonds between Der p 1 and VH of 10B9 involve, respectively, Ala12 and Tyr53, Glu13 and Arg98, Asp15 and Tyr107,
Asn179 and both Ser31 and Gly32, Gly182 and Ser31, and Asp198 and
Tyr106 (Table III). Gln181 was found to form a hydrogen bond with
Ser31 in only one of the copies of the Der p 1–10B9 complex.
There is a possibility that two Der p 1 residues form cation–p
interactions—Gln18 with Tyr107 and Arg17 with Tyr106 of the VH
of 10B9—but a higher resolution structure of Der p 1–10B9
complex would be required to verify these interactions.
Relative location of the epitopes for the mAbs 10B9 and 4C1
on Der p 1
Phe100, Leu101, Thr102, Thr103, and Asn105, which adopt a welldefined a helical conformation (one and a half turn a helix as
shown in Supplemental Fig. 1B). The loop placement during the
binding of Der p 1 is shown in Supplemental Fig. 1A. The overall
conformation of the Fab fragment of mAb 10B9 is not significantly changed, in comparison with the uncomplexed form, but
rather all the CDR regions are shifted upon binding to Der p 1.
There are two salt bridges formed by Der p 1 and 10B9. The first
is formed by Asp198 and Arg53 of the VL and the second is formed
between Glu13 and Arg98 of the VH. The asymmetric unit contains
two copies of Der p 1–10B9 complex; 14 hydrogen bonds have
been identified in the first copy and 13 in the second copy of the
complex. The hydrogen bonds between Der p 1 and VL of 10B9
involve the following residues: Gln18 of Der p 1 and both Gly91
and Asn92 of 10B9 (asparagine forms a hydrogen bond only in one
of the copies of Der p 1–10B9 complex), Asp198 of Der p 1 and
Arg53 (as well as Tyr106 of VH) of 10B9,and Asn199 of Der p 1 and
Arg53 of 10B9. Arg20 was found to form a hydrogen bond with
Ser30 of the VL, but only in one of the copies of the complex. The
Table II. Hydrogen bonds formed by Der p 1 with the Fab fragment of
mAb 5H8 (4PP1)
5H8
Der f 1
Der p 1
Arg52
Arg51
VL
VH
Asp103
Gly91
54
53
Thr
Ser55
Gln85
Ser94
Ser109
Gln110
His111
Gln
Ser54
Gln84
Tyr93
Ala108
Gln109
Arg110
Gly113
Ile114
Ser115
Gly112
Ile113
Ser114
Met223
Leu222
58
Asp
Arg56
Arg101
Thr30
Gln100
Gly53
Gln100
Tyr102
Asp103
Arg101
Tyr32
Tyr50
Lys92
Distance (Å):
Chains A/B
3.0/2.9
2.7/3.0
2.5/2.7
2.9/3.0
2.6/2.4
2.5/2.6
3.2/3.1
2.9/3.1
2.7/2.7
2.6/2.6
3.2/3.1
2.5/3.2
2.9/3.0
3.1/3.0
2.4/2.8
The residues in Der f 1 that correspond to the Der p 1 residues that form the 5H8
binding epitope are shown to highlight the differences between both allergens. Hydrogen bonds were found by PISA and Molecular Operating Environment. Residues
creating hydrogen bonds are in rows.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. (A) The relative position of Der p 1, and the Fab of 5H8,
4C1, and 10B9. The Fab fragments of the Abs are colored as follows: 4C1,
blue; 10B9, red; and 5H8, orange. Der p 1 is represented as a green surface
with epitopes binding each Ab colored in a similar color to the corresponding Ab. The overlapping area of the 4C1 and 10B9 binding epitopes
is purple. The surface of Der p 1 highlighting the epitopes for the Fab
fragments of mAb 4C1 (B and E), 10B9 (C and F), and 5H8 (D and G) is
shown. The epitope binding the Fab fragment of mAb 4C1 is colored in
pink (the part of the epitope where the VH of the Fab fragment of mAb 4C1
is bound is colored dark pink, the part where the VH and VL of the Fab
fragment of mAb 4C1 are bound is colored light pink), the part of epitope
where the Fab fragment of mAb 10B9 is bound is colored orange (VH),
light orange (VH and VL), and yellow (VL only). The epitope binding 5H8
is colored as follows: VH, dark cyan; VH and VL, blue; and VL, cyan. (E)–
(G) represent the structural conservation between Der p 1 and Der f 1
calculated with VMD.
Even though the epitopes for both 4C1 and 10B9 largely overlap,
the relative position of the Abs is not shifted, but rather the mAb
4C1 binds to its epitope in a position that is “rotated counterclockwise” by a little more than 90˚ with respect to the position
assumed by 10B9 upon binding to its epitope on Der p 1 (Fig. 1B,
1C). The residues of Der p 1 that participate in hydrogen bonding
with both 10B9 and 4C1 are Glu13, Arg17, Gln18, and Asp198.
Residues specific to hydrogen bond formation with 4C1 are Arg20
and Arg156, whereas Ala12, Asp15, Gln181, Gly182, Tyr185, and
Asn199 participate in hydrogen bonding with 10B9. A surfaceexposed tyrosine residue of the Fab fragment of mAb 4C1
forms hydrogen bonds with an arginine residue on the surface of
the Ag, which is observed at the Der p 1 and 4C1 interface
(Supplemental Figs. 3A, 3B, 4A, 4B as well as our previous work
in Ref. 10). These bonds are not observed in the Der p 1–10B9
complex. However, hydrogen bonds are present between a surface
tyrosine on the Ab and an aspartate residue on the surface of
Der p 1. Furthermore, such hydrogen bonds are not present at
the interfaces formed by the Fab fragments of mAb 4C1 or 5H8
and Der p 1. The interface between 10B9 and Der p 1 comprises
several hydrogen bonds that are formed between serine and arginine as well as asparagine/glutamine and glycine. In the 4C1
complex, however, the interface is formed between arginine, aspartate, serine, or threonine from 4C1 and tyrosine, arginine, aspartate,
glutamine, or glutamate on Der p 1, with the most abundant linkage
being between tyrosine from 4C1 and arginine from Der p 1 (figure
3a of Ref. 10).
There is a conformational change of the Der p 1 loop from
residue 179 to 185 (root mean square deviation of 0.7 Å, where
The Journal of Immunology
311
Table III. Hydrogen bonds formed by Der p 1 with the Fab fragment of
mAb 10B9 (4PP2)
10B9
Der f 1
Der p 1
Ser13
Glu14
Ala12
Glu13
Asp16
Ser19
Asp15
Gln18
Arg20
Ser180
Arg20
Asn179
Gln182
Gly183
Asp199
Gln181
Gly182
Asp198
Ser200
Asn199
VL
VH
Tyr53
Arg98
Arg98
Tyr107
Gly91
Asn92
Ser30
Ser31
Gly32
Ser31
Ser31
Tyr106
Arg53
Arg53
Arg53
Distance (Å):
Chains E/F
2.7/2.6
2.9/3.0
2.8/2.7
2.8/2.7
3.3/3.2
3.3/3.5
2.9/NA
2.6/2.7
3.0/2.8
3.6/3.3
2.8/2.8
2.7/2.8
2.7/3.0
3.0/3.2
2.5/2.7
overall root mean square deviation is 0.6 Å) calculated between
Der p 1 complexed with 4C1 and Der p 1 complexed with 10B9.
This conformational change, however, is not observed between
uncomplexed Der p 1 and the Der p 1–10B9 complex. This loop
provides strong interactions with the paratope on the H chain of
the Fab fragment of mAb 10B9, but it is on the edge of the epitope
and does not form any substantial interactions with the L chain of
the Fab fragment of mAb 4C1 (Supplemental Fig. 1A). Therefore,
the conformational change might be induced upon binding of
water molecules between the 4C1 Ab and Der p 1, which would
therefore create bridging interactions between Asn179, Ser180, and
Tyr186 with Glu106 of CDR H3 (as described in Ref. 10). However,
this rearrangement is located on a peripheral part of the Der p 1–
4C1 interface, and other variables might contribute to the rearrangement.
Comparison between Der p 1 allergen epitopes for 10B9, 4C1,
and 5H8 Abs and the corresponding surface on Der f 1
The interfaces created between Der p 1 and 10B9 or Der p 1 and
5H8 (both species-specific mAbs) have larger contact areas than do
the interfaces between Der p 1 and 4C1 and Der f 1 and 4C1 (crossreactive mAb) (∼910 and ∼845 Å2 versus ∼745 and ∼760 Å2,
respectively; calculated by PISA). As mentioned previously, both
10B9 and 5H8 are species specific, whereas the 4C1 Ab is crossreactive between Der p 1 from D. pteronyssinus and Der f 1 from
D. farinae. The deletion of serine in the eighth position of Der p 1
causes an offset by one residue in the amino acid numbering between Der p 1 and Der f 1, and thus Der f 1 numbering of amino
acid residues is used in comparisons of Der f 1 with Der p 1 for
both allergens in this section.
The QH algorithm was used as the scoring function on structures
superimposed in VMD. The calculated QRES values were assigned
for every residue, and the surface was marked in such a way that
the amino acids with side chains that had the most similar orientation and shape were colored blue through white to red (which
indicates that the orientation and shape of the side chains were
different) (Fig. 1E–G).
The 4C1 Ab is cross-reactive between two dust mite allergens,
Der p 1 and Der f 1, whereas 10B9 is specific for Der p 1. To explain
this observation, we compared the residues of Der p 1 that form
FIGURE 2. The 10B9 (A and C) and 5H8 (B and D) binding epitopes on
Der p 1 are shown. The amino acids that form hydrogen bonds between
Der p 1 and Abs are colored as follows: Der p 1, shades of green; 10B9,
purple; and 5H8, blue. The labels showing residue names and numbers are
colored in the same way. The amino acid residues in Der f 1 that correspond to the residues of the 10B9 epitope on Der p 1 are shown in (C) and
colored purple, and 5H8 is shown in (D) and colored orange. The cyancolored residues shown in (C) and (D) come from Der f 1 and correspond
to residues from Der p 1, labeled purple in (C) and orange in (D).
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The residues in Der f 1 that correspond to the Der p 1 residues that form the 10B9
binding epitope are shown to highlight the differences between both allergens. Hydrogen bonds were estimated by PISA and a cutoff of 3.3 Å was chosen. Residues
creating hydrogen bonds are shown in rows. Underlined amino acids also form salt
bridges.
interactions with 10B9 to corresponding residues from Der f 1. The
residues that form the 10B9 binding epitope on Der p 1 are partially
conserved in Der f 1. In addition to the surrounding amino acids not
directly involved in the interaction with 10B9, the major differences between Der p 1 and Der f 1 in the 10B9 binding epitope
include a substitution of residues Ser13, Ser19, Ser180, Thr181, and
Ser200 in Der f 1 by Ala, Gln, Asn, Ala, and Asn, respectively, in
Der p 1 (Figs. 1F, 2C), which allows for hydrogen bond formation
between Der p 1 and 10B9 (Table III). However, a lack of these
hydrogen bonds is not the major factor preventing the binding of
10B9 to Der f 1. A comparison of the available Der p 1 and Der f 1
structures reveals that there is a significant difference in conformation and composition of the 179–183 region (Asn-Ala-Gln-GlyVal) of Der p 1 and corresponding 180–184 fragment (Ser-ThrGln-Gly-Asp) of Der f 1. The different conformations of these
regions results in a significant shift of the central Gln position,
with corresponding Ca atoms being shifted by ∼3 Å. This shift
results in Gln181 in Der p 1 being “parallel” to the allergen’s
surface, whereas in the case of Der f 1, this residue is “perpendicular” to the allergen’s surface. A superposition of the Der p 1–
10B9 complex and Der f 1 shows that the perpendicular conformation of Gln182 in Der f 1 results in a steric clash with Ser31 from
the H chain of the Ab. The density map, which is of good quality
around Gln181, allows for the assumption that a small change in
the amino acid composition results in different conformations of
this region in Der p 1 and Der f 1.
The 5H8 binding epitope area on Der p 1 is mostly conserved in
Der f 1 (15 of 24 aa residues), but there are several differences
between them (Table II). Residues Thr54 and Ser94 of Der f 1 are
substituted by Gln and Tyr, respectively, in Der p 1, thus making it
impossible for Der f 1 to form hydrogen bonds with the Fab
fragment of mAb 5H8 (Fig. 2D). Der f 1 amino acid residues
Thr54, Glu91, Ser94, Ser109, and Tyr112 are substituted by Gln, Gln,
Tyr, Ala, and Gly residues, respectively, in Der p 1, which creates
a nonconserved spot in the central part of the 5H8 epitope surface
312
ANTIGENIC STRUCTURE OF Der p 1
area (white spot in the central part of Fig. 1G) and therefore
changes the shape of the surface of the 5H8 binding epitope
(Fig. 2D). Some of the amino acids in Der f 1 (Gln86, His111, and
Met223) that correspond to the 5H8 binding epitope on Der p 1 are
substituted (His, Arg, and Leu), but in the Der p 1–5H8 complex
all the interactions are mediated by the main chain atoms for
these residues and the side chains do not create sterical clashes.
Therefore, these residues should not interfere with the possibility
of 5H8 binding to Der f 1.
Analysis of Ag–Ab interactions in complexes reported in PDB
Table IV. Ratios between the amino acid occurrences observed in the interfaces between Ags and Abs versus
expected amino acid occurrences
Ags
Interface
Amino Acids
Proteins
Peptides
Abs
Abs
Der p 1
10B9
5H8
Abs
4C1
Proteins
10B9
Peptides
5H8
4C1
Der p 1
Values were calculated from a frequency of occurrences of a particular amino acid in all surfaces of nonredundant Fab–protein or
Fab–peptide complexes available in the pdbaa National Center for Biotechnology Information’s BLAST database as of 2013. Darker
intensity indicates a higher ratio.
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The methods used to investigate Der p 1 complexes with Abs made
it possible to not only analyze these complexes, but all nonredundant complexes of Fab fragments of Abs with proteins or
peptides available in the PDB of resolution $3.0 Å (n = 314 as of
2013). The particular amino acids found in the protein–Ab or
peptide–Ab interfaces are shown in Supplemental Fig. 3G and 3I.
However, the observed frequency of the amino acids on the surface of the epitopes, in general, is very similar to the expected
frequency of these amino acids on the surface of proteins from the
dataset, with charged or polar residues more frequent and hydrophobic residues less frequent. Serine and threonine are the
exception, however, with their occurrence being essentially 2-fold
less frequent in epitopes than in other parts of the surface of
proteins from the dataset. Glutamine residues in the epitopes are
approximately one third more frequent than expected. The observed frequency of the amino acids in the paratopes differs significantly from the expected frequency, with tyrosine being 6-fold
more frequent and tryptophan being 3-fold more frequent in the
paratope interfaces than on the surface of proteins from the dataset
(Table IV). The observed value of serine frequency in the paratopes of Fab–protein complexes is almost equal to the expected
serine frequency on the protein’s surface (Table IV). This
frequency of serine is not observed in the paratopes of the Abs
interacting with peptides and is half of the expected value
(Supplemental Fig. 3J, Table IV). Among charged amino acids,
only arginine and histidine are more frequent than expected in the
paratopes of proteins than on the surface. The frequency of lysine
is lower than expected for protein–Ab complexes or almost equal
to the expected value for peptide–Ab complexes. We have also
analyzed amino acids from epitopes and paratopes with respect to
their contribution to the interface area; this analysis is detailed in
the Supplemental Fig. 4.
Supplemental Fig. 4 shows the area contributed to the interfaces
by particular amino acids. The amino acids found in the epitopes
of proteins and peptides generally contribute more area to the
interfaces than amino acids found in the paratopes. Residues that
contribute the most area to the protein–Ab interfaces found in the
epitopes are arginine, histidine, lysine, methionine, phenylalanine,
and tryptophan, whereas the residues that contribute the most area
to the protein–Ab interfaces found in the paratopes are arginine,
methionine, phenylalanine, and tryptophan. The amino acids found
in the epitopes of the peptide–Ab interfaces contribute even more
area to the interface as compared with the protein–Ab interface, but
this observation can be explained by the small size of peptides. In
the paratopes of the peptide–Ab interfaces, methionine contributes
The Journal of Immunology
the most area. Curiously, and in contrast with the larger area
contribution of methionine in all of these interfaces, the observed
frequency of methionine, found in both the epitopes and paratopes, is half the expected frequency of methionine found on the
surface of proteins in the dataset, with the exception of methionine
found in the peptide epitopes (Table IV).
Amino acid composition of the interface of the complexes of
Der p 1 with Abs
FIGURE 3. Hydrogen bonds formed
between (A) Der p 1 and the Fab fragment
of mAb 10B9 (two copies from asymmetric unit), (B) Der p 1 and the Fab
fragment of mAb 5H8 (two copies from
asymmetric unit), (C) all nonredundant
structures of complexes of proteins with
Fab fragments of mAbs, and (D) all nonredundant peptides with Fab fragments of
mAbs are shown. The complexes of proteins and peptides used for analysis were
solved at a resolution $3.0 Å. The number of hydrogen bonds between particular
amino acids is shown in grayscale (on
right) where the higher the number of
hydrogen bonds, the darker the square.
Abs are on the x-axis, and Ags are on the
y-axis of each plot.
physicochemical similarity to glutamine, is only present in the
paratope of the 4C1 Ab in twice the expected frequency, and is
absent in the 10B9 and 5H8 paratopes. A rather unusual observation, in comparison with other Abs, is that the paratope of the
4C1 Ab contains 8-fold more arginine than the usual arginine
frequency on protein surfaces, and it is much higher than the
general arginine content in the paratopes, as well as in the paratopes of 10B9 and 5H8 (Table IV).
The hydrogen bonds formed between glycines of the Fab
fragment of mAb 5H8 or the Fab fragment of mAb 10B9 and the
Der p 1 allergen are quite common in comparison with the hydrogen bonds created between all Abs and all proteins or peptides
(Fig. 3). The differences between amino acids involved in hydrogen bonds between the paratopes of the Fab fragments of
mAbs 4C1, 10B9, and 5H8 and Der p 1 allergen are significant.
Most of the hydrogen bonds in the paratope of 10B9 are formed
by serine with arginine, asparagine, glutamine, or glycine and by
tyrosine with alanine or aspartate on Der p 1 (Fig. 3A). Hydrogen
bonds on the 5H8 paratope are formed by aspartate with arginine,
glutamine, or glycine, by glutamine with arginine or asparagine,
and by tyrosine with serine or arginine on the surface of Der p 1
(Fig. 3B). Quite rare hydrogen bonding interactions can be observed at the Der p 1 epitope and 5H8 paratope interface. Namely,
the hydrogen bonds are created between the main-chain atoms of
Ile83 and Ile113 of Der p 1 and side chain of Arg101 from 5H8.
Additionally, Ile113 from Der p 1 and Asp103 from 5H8 also form
hydrogen bonds. Surprisingly, there are also not common hydrogen bonds at the interface between Der p 1 and 5H8—formed
between glutamines from Der p 1 and arginines and aspartates
from 5H8 Ab.
The amino acid composition of the Der p 1 epitopes that bind to
the Fab of mAb 10B9, 5H8, and 4C1 does not contain all of the
standard amino acids. The amino acids that are missing or un-
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The interface area of Der p 1 complexed with the Fab fragments of
mAb 4C1, 10B9, or 5H8 (Supplemental Figs. 3, 4) is composed of
almost every charged or polar amino acid (tyrosine, threonine,
arginine, serine, glutamine, aspartate, glutamate, and aspartate).
However, hydrophobic amino acids such as leucine and isoleucine
are also present. The amino acids that form the interface, which
are found in any of the three Abs, include mostly tyrosine, arginine, threonine, and serine. Tyrosine is one of the main amino
acids that contribute the most surface area to the interfaces in both
the epitopes of Der p 1 and the paratopes of the Abs. The number
of tyrosines is approximately 5- (5H8) to 10-fold (10B9 and 4C1)
higher in the paratopes than the average tyrosine frequency on
protein surfaces of Fab–protein and Fab–peptide complexes
(Table IV). Arginine is significantly more abundant in the epitopes
of Der p 1 and the paratopes of all three Abs. In the paratope of the
Fab fragment of mAb 4C1, arginine is 8-fold more frequent than it
is on protein surfaces of Fab–protein and Fab–peptide complexes
(Table IV). The interface area on the Der p 1 epitopes contains
a significantly higher frequency of arginine, aspartate, glutamate,
and glutamine than the general frequency of these amino acids on
the surface of the proteins from the dataset. Arginine is 4- to 6fold more frequent, aspartate is 3- to 4-fold more frequent,
glutamate is 2-fold more frequent, and glutamine is 2.5- to 4-fold
more frequent than what is expected. Asparagine, despite the
313
314
derrepresented are mostly nonpolar; however, some polar or
charged amino acids may not be present in every epitope. Aspartate
and glutamate are not present in the 5H8 binding epitope, but they
are very common in the 4C1 and 10B9 binding epitopes. Serine is
absent in the 4C1 binding epitope, but it is present in other epitopes.
The 4C1 binding epitope contains threonine, which is absent in
both 5H8 and 10B9 binding epitopes. Valine and lysine are absent
in all of the three epitopes. Alanine is overrepresented in the 4C1
and 10B9 binding epitopes and is present in equal amounts to the
expected value in the 5H8 binding epitope on Der p 1. The content
of isoleucine, despite being a nonpolar amino acid, is significantly
higher than expected in the 4C1 and 5H8 epitopes, but isoleucine is
absent in the 10B9 epitope. Even though Der p 1 epitopes that bind
4C1 and 10B9 mostly overlap, their amino acid composition is
different. The epitope that binds 10B9 contains glycine and serine
in lower than expected quanitities and histidine in higher than the
expected amount. However, it does not contain either threonine or
isoleucine, both of which are present in the 4C1 binding epitope.
The comparative analysis of interfaces formed by Der p 1 with the
Fab fragments of mAb 4C1, 10B9, and 5H8 required an investigation of the content of the interfaces and hydrogen bonds formed
by all Fab fragments of Abs complexed with proteins or peptides.
Analysis of Ag–Ab interfaces showed (Supplemental Figs. 3, 4)
that tyrosine is found .5-fold more often on the surface of the
paratopes than on the surface of proteins in general (Table IV). It
strongly suggests the important structural role of tyrosine in
forming interfaces and its presence in interface areas found in all
complexes of Fab fragments of Abs with proteins, peptides, or
nucleic acids (unpublished data) and it is consistent with earlier
studies (40). Tryptophan, which shares some of the properties of
tyrosine, is found to be three times more frequent on the surface of
paratopes than on the surface of proteins in general. A higher
concentration of aromatic amino acids at the binding interfaces is
consistent with a previous report (41). The quantitative analysis of
the binding interfaces showed that aromatic amino acids found in
paratopes tend to contribute significantly more surface area to the
interfaces than do other amino acids found in paratopes, with the
exception of methionine. Methionine, despite its very low frequency of occurrences in Ag–Ab interfaces, usually contributes
a significant surface area to the interface when present. Serine and
threonine, despite having polar side chains, are found to be 2-fold
less frequent in the protein–Ab or peptide–Ab interfaces, as in
other parts of proteins. The observed frequency of serine in the
paratopes, however, is almost equal to the expected value.
Discussion
Most human IgE Abs (.80%) are directed against cross-reacting
epitopes on Der p 1 and Der f 1 (42, 43). The mAb analyzed,
including the cross-reactive mAb 4C1 and species-specific mAb
10B9 and 5H8, partially inhibited IgE Ab binding to Der p 1 (11,
42, 44). The three epitopes identified here on Der p 1, by x-ray
crystallography, provide the basis for an ongoing analysis of antigenic determinants involved in IgE Ab binding.
The unique a helical loop in CDR H3 of 10B9 Ab, which is able
to fit into a cavity on Der p 1’s surface, is formed by a conformational change that occurs upon binding to Der p 1. The a helix
in CDR H3 is present in ,20 out of ∼1000 (∼2%) Ab structures
reported to the PDB. This form of secondary structure is very rare,
and its role has been described as inhibitory (it competes for
binding site by mimicking the structure of the other protein) (45),
as affecting Ag recognition (46, 47), or it can form a tentacle that
provides a large interaction surface for Ag binding (47). In almost
all other structures of Fab fragments with Abs, the CDR H3 forms
a random coil loop. The function of the CDR H3 loop of 10B9 can
be explained as affecting Ag recognition and providing a backbone for the formation of the Der p 1-10B9 interface, by increasing the size of the paratope area and participating in forming
hydrogen bonds. The interaction between 10B9 and Der p 1 is an
example of “induced fit” protein interactions rather than “lockand-key.” Considering the lock-and-key interaction of Der p 1
with 4C1 Ab described previously (10), this study shows that both
types of Ag–Ab interactions can occur even within a similar area
on the Ag surface.
The Der p 1 epitope binding 5H8 Ab may be affected by sequence polymorphisms (48) in a few sites (Ala108 and Ile113).
However, structural analysis reveals that these amino acids are
involved in the interface creation by their main-chain atoms, and
their variance most likely does not interfere with 5H8 binding.
Both 10B9 and 4C1 epitopes do not contain residues that are associated with sequence polymorphisms (48). A comparison of mature
pro-rDer p 1–Ab complex structures with the structure of pro–Der
p 1 clearly indicates that the presence of the propeptide does not
interfere with the binding of 4C1, 10B9, and 5H8 Fab fragments
to the allergen.
Structural studies of Der p 1 and Der f 1 allergens allowed us to
directly compare the epitopes that bind the Fab fragments of mAb
4C1, 10B9, or 5H8. Such a comparison provides detailed insight, at
the molecular level, about which of these allergen surfaces are
responsible for the fact that 10B9 and 5H8 Abs are Der p 1 specific,
whereas 4C1 is cross-reactive and binds both Der f 1 and Der p 1
(10). The results in the present study indicate which of the Der p 1
amino acids, responsible for binding 10B9 and 5H8 Abs, are
different between Der p 1 and Der f 1, rendering the latter impossible to bind 10B9 and 5H8. Residues that were different between 10B9 and 5H8 binding epitopes on Der p 1, as well as the
corresponding surface area of Der f 1, are mostly charged and
polar. However, there are a few substitutions of nonpolar amino
acids such as alanine to small polar amino acids such as threonine
and serine that may affect their attractions with the amino acids
found in the paratopes of 10B9 and 5H8 Abs and therefore contribute to species specificity. In Der f 1, the number of amino acids
that differed from the corresponding residues that form hydrogen
bonds between 10B9 or 5H8 and Der p 1 was significant. These
substitutions apparently contributed to the lack of binding of these
two Abs to the Der f 1 allergen, despite a high sequence identity
(∼75% at the surface level) and similarity (∼83% at the surface
level) between these two allergens. These results indicate that
relatively small differences in the allergen surface may impair Ab
binding. For example, such changes may involve the presence of
a few nonconserved residues, which were observed in the case of
the Der f 1 region that corresponds to the 5H8 epitope in Der p 1.
However, these differences may be more subtle as it occurs on the
surface of Der f 1, in the region corresponding to the 10B9 epitope
in Der p 1. In this case, Der f 1 has smaller residues than the
corresponding residues in Der p 1, and it therefore cannot form
many binding interactions with 10B9. Additionally, to the “missing”
interactions, the conformation of the 180–184 fragment (Ser-ThrGln-Gly-Asp) in Der f 1 is not compatible with 10B9 binding, as the
main chain of Gln182 generates a steric clash between the allergen
and the Ab.
The amino acid composition of the Der p 1 epitopes for 4C1,
10B9, and 5H8 Abs demonstrates that in general there is little
preference for particular amino acids. However, all polar or charged
amino acids are much more frequent than those that are nonpolar.
The surface of the paratope is built from polar amino acids such as
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Amino acid analysis of complexes of proteins or peptides with
Fab fragments of mAbs
ANTIGENIC STRUCTURE OF Der p 1
The Journal of Immunology
into account the surface contributed to the interface area by particular amino acids. The information obtained from the contribution of
surface area and preferences in creating hydrogen bonds between
Ags and Abs may be used alongside sequences and structures as
an element of creating new epitope prediction tools with very
high prediction accuracy.
The determination of the three-dimensional structure of allergen–Ab complexes reveals conformational epitopes, which are the
main epitopes in inhalant allergens, such as Der p 1 (58). This
approach for mapping Ab epitopes represents a major advantage,
compared with previously used strategies, which are based on the
identification of Ab-binding peptides that only contain linear
epitopes. Therefore, the results of our structural studies of group 1
mite allergens may be used not only to design experiments
allowing for the identification of IgE binding epitopes, but also to
design recombinant versions of allergens for application in immunotherapy (2).
Disclosures
A.P., J.G., L.D.V., and M.D.C. are employees of Indoor Biotechnologies, Inc.,
and M.D.C. is also founder and co-owner of the company. The work in the
present study was sponsored by a multiple principal investigator R01 grant to
Indoor Biotechnologies, Inc. (principal investigators A.P. and M.D.C.) in collaboration with the University of South Carolina (principal investigator M.C.)
and the University of Virginia. The remaining authors have no financial
conflicts of interest.
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tyrosine or serine, possibly to increase sterical compatibility and to
compensate for the epitope’s charge. In contrast to 10B9 and 5H8,
the paratope of the 4C1 Ab has a very high arginine content, which,
by adding a positive charge to the Der p 1 binding paratope, may
partially contribute to its cross-reactivity.
Analysis of Der p 1 and Der f 1 allergen–Ab complexes
prompted us to investigate all protein–Ab and peptide–Ab complexes that have their structures determined. We have developed
a unique methodology that allows analysis of the properties of
epitopes and paratopes as: relative frequency of amino acids, areas
of interactions for particular amino acids, and hydrogen bonding
interactions. This methodology allows for the generation of plots
highlighting diverse aspects of epitopes and paratopes. The
analysis of the amino acid composition of the interfaces in the
structures deposited to PDB (Table IV, Supplemental Fig. 3)
showed that none of the amino acids is significantly overrepresented in the epitopes. However, amino acid composition of the
epitopes shows bias toward polar (with the exception of serine and
threonine) and charged amino acids. Such bias is observed for
both proteins and short peptides. The observed frequency of serine
and threonine is half of the expected value. One could expect such
results for proteins, taking into account that the residues forming
epitopes are on their surface, but this trend is not so obvious for
peptide–Ab complexes. The amino acid composition of the paratopes is also highly biased toward polar (except for serine and
threonine) and charged amino acids with significant absence of the
nonpolar amino acids. It is also striking that tryptophan and
especially tyrosine residues are significantly overrepresented in
CDRs of the Abs. A special role of the tyrosine was already noticed (40) and most likely could be explained by the chemical
nature of this amino acid that allows it to participate in many types
of bonding interactions.
The analysis of the Ag–Ab interfaces revealed a preference for
certain amino acids that are involved in creating hydrogen bonds
between Abs and Ags. The amino acids that are most often involved in hydrogen bond formation on the surface of the paratopes
are tyrosine (22%), serine (14%), aspartate (13%), arginine (10%),
and asparagine (9%). On the surface of the epitopes, most of the
hydrogen bonds are created by arginine (15%), glutamate (11%),
aspartate (10%), lysine (10%), serine (9%), and asparagine or
glutamine (both are 8% each). A closer look at the surface area of
the Ag–Ab interfaces indicates that the number of amino acids is
lower for the Ags and higher for the Abs. The relative area contributed to the interfaces by particular amino acids, however, is
quite the opposite, being higher for the Ags and lower for the Abs.
This is consistent with the fact that, in general, Abs must have the
possibility to evolve and quickly adjust to recognize a wide variety
of Ags (clonal selection and affinity maturation). Having several
residues in the binding site provides greater flexibility to precisely
recognize different epitopes. Such variability is not needed at the
level of the epitopes for the immune response to be flexible. It may
not even be desired in the design of molecules that no longer bind
the Ab, as the mutation of just a few residues, which contribute
a large surface area, might be enough to prevent undesired recognition by Abs.
There are various methods that currently exist for the prediction
of linear (49–51) and conformational (52–55) epitopes. The linear
epitope prediction algorithms do not provide enough value, as
most of the epitopes in globular proteins are discontinuous or
nonlinear and brought together during protein folding (56, 57).
The algorithms used in these methods take into account various
properties of the epitopes such as the hydrophilicity scale, protein
secondary structure, and protein flexibility, but their accuracy is
usually ∼80%. There is no epitope prediction algorithm that takes
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ANTIGENIC STRUCTURE OF Der p 1