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(Received for publication, January 22, 1996; and in revised form, March
8, 1996) From the
The three-dimensional structure of the Fab of TR1.9, a
high-affinity IgG1,
The effector mechanisms in human autoimmune diseases may involve
either T cells or B cells. Presently accepted examples of T
cell-mediated autoimmune disease are diabetes mellitus type I and
multiple sclerosis. On the other hand, autoantibodies to the
acetylcholine receptor are responsible for myasthenia gravis and
autoantibodies to the thyrotrophin receptor cause the hyperthyroidism
of Graves' disease. The most common organ-specific autoimmune
disease in humans is Hashimoto's thyroiditis. IgG class
autoantibodies to thyroid peroxidase (TPO), ( One of these
recombinant TPO Fabs, TR1.9(5) , interacts with the B2 domain
in the immunodominant region. Like other IgG class autoantibodies,
TR1.9 binds specifically to its antigen and it is encoded by genes
which appear to be somatically mutated from the germline (reviewed in (10) ). In contrast, IgM class autoantibodies are frequently
polyreactive and may be derived from unmutated or only slightly mutated
germline genes (see for example, (11) ). Information on the
three-dimensional structure of human TPO-specific Fab and, ultimately,
the Fab-TPO complex will provide insight into TPO recognition by the
immune system. In this report, we present the crystallographic
analysis, at 2.0-Å resolution, for TPO-specific Fab TR1.9. Of the
limited number of human antibodies for which crystal structures have
been determined(12) , none are autoantibodies. The present
data, therefore, present the first structural analysis of a human, IgG
class, disease-associated autoantibody.
Figure 1:
Data completeness
plotted against resolution for all non-zero reflections and for those
with F
A
2F
nued for
the time being.
Figure 2:
Stereodrawing of a portion of the
2F
The structure was then refined using X-PLOR with
data for which F Another round of model rebuilding using FRODO was performed based on
2F From this point onward, all the data in
the resolution range 10-2.0 Å, for which F The average B-factor is 30.9 Å
squared (r.m.s.d. = 16.7) for the 439
Figure 3:
B-factor plots for the two chains in
TR1.9 Fab: solid, main chain; dotted, side chain
(zero side chain values correspond to glycines). The residue numbers
are sequential.
The final model has 3318
protein atoms comprising 214 amino acids in the light and 225 amino
acids in the heavy chain, plus 216 water molecules. The root mean
square deviation from ideality is 0.016 Å for bond lengths, and
3.5 and 1.4 degrees for bond and dihedral angles, respectively. Four
non-glycine residues have
Figure 4:
Ribbon drawing of TR1.9 Fab showing the
light chain on the left and the heavy chain (darker) on the right. The
variable domains are at the top and the constant domains of the Fab are
at the bottom. The molecular surface, shown as dots, covering
the CDR residues was computed using program MS of Connolly (27) ; a probe radius of 1.7 Å was
used.
The
molecular surface (27) that covers the CDRs of TR1.9 is
included in Fig. 4. The CDR surface of TR1.9 is revealed to be
relatively flat. Other antibodies to intact protein antigens also have
relatively flat CDR surfaces, in contrast to antibodies to haptens and
other smaller ligands which display pronounced grooves or pockets in
their CDR surfaces(28) . Results from the crystallographic
analysis of many antibody-ligand complexes strongly suggest that the
combining site of an antibody is primarily constructed with CDR
residues, although on rare occasions neighboring framework residues
have been found to be involved also. Thus the CDR surface of TR1.9 most
probably portrays the topography of its combining site. The relative
flatness of the surface implies that the epitope for TR1.9 on TPO is in
the main also flat. The CDRs of TR1.9 are canonical: CDR1-L belongs
to the canonical group 2, CDR2-L to group 1 (the only group identified
so far), CDR3-L to group 1, CDR1-H to group 1, and CDR2-H to group 3;
no canonical groups have been identified for CDR3-H(29) .
Figure 5:
Stereodrawing of the
Figure 6:
Stereodrawing of the
The TR1.9 V The C A
greater variation is observed for the human V The comparison of the human C In this collection,
the immunoglobulin Mcg is found to be the most different, not only in
V
Ignoring the
differences at the NH Relative to the closest
germline, five somatic mutations appear to have occurred in TR1.9
V
Figure 7:
Ribbon drawings of the Fv of TR1.9 viewed
from the side (top) and end-on (bottom). V
The insertion of an extra residue in the NH
Some other replacements may contribute to the high
affinity of binding. Of the 15 putative somatic mutations that appear
to have occurred in the maturation of TR1.9, four involve asparagines.
Three of those are in CDRs (at position 53 in CDR2-L and at positions
52 and 54 in CDR2-H) and the fourth is at the framework position 20 in
the light chain. It has been noted that asparagines in CDRs frequently
form hydrogen bonds with main chain atoms, apparently stabilizing the
conformation of the local structure(43) . In TR1.9, the
asparagines at positions 20 and 53 in the light chain are exposed to
solvent and do not form hydrogen bonds, so that they are probably not
critical to conformational stability. The two other somatic changes
involving asparagines occur at positions 52 and 54 in CDR2-H, where
asparagines in the closest germline V The
structural basis for the high affinity will be clarified by the crystal
structure of the complex of TR1.9 with TPO. Knowledge of the structural
details of the binding of TR1.9 to TPO will add to our understanding of
TPO recognition by the immune system, including antigen presentation by
TPO-specific cells, and will provide new insights into humoral
autoimmune diseases in humans.
The atomic coordinates and structure factors (1VGE, R1VGESF)
have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12191-12198
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CRYSTAL STRUCTURE OF A THYROID PEROXIDASE AUTOANTIBODY FAB (*)
human autoantibody to thyroid peroxidase, was
determined crystallographically to a resolution of 2.0 Å. The
combining site was found to be relatively flat, like other antibodies
to large proteins. Sequence differences from the most closely related
germline genes mainly occur at positions occupied by residues with
outward-pointing side chains. An increased deformability of the second
and third complementarity-determining regions of the heavy chain may
result from the replacement of two germline asparagines and the
presence of several glycines, and may allow ``induced fit''
in the binding to antigen. Four exposed charged residues, resulting
from the use of a particular D (diversity) and J (joining) segments in
the assembly of the heavy chain, may contribute to the high affinity of
antigen binding. The crystal structure of TR1.9 Fab is the first for a
human IgG high-affinity autoantibody.
)a large
glycoprotein (107 kDa) expressed on the apical surface of thyroid
cells, are an invariable marker of the disease and may contribute to
thyroid damage and hypothyroidism (reviewed in (1) ). Recently,
we have generated a panel of 42 human monoclonal TPO autoantibodies
(expressed as Fab) from thyroid-infiltrating plasma cells by screening
immunoglobulin gene combinatorial libraries with eukaryotic recombinant
TPO(2, 3, 4, 5, 6, 7) .
These recombinant IgG class Fabs have a high affinity (K
10
M) for TPO and recognize overlapping conformational
epitopes in a restricted region of the
molecule(5, 8, 9) . Furthermore, the TPO Fabs
compete for binding to TPO by >80% of autoantibodies in serum from
most patients and, consequently, they define a TPO immunodominant
region(3, 5, 8, 9) .
Expression of TPO-specific Fab TR1.9
To permit
higher levels of expression, the heavy and light chain genes for TR1.9
in the Immunozap vector (5) were subcloned in the XhoI
and XbaI sites of pBP101(13) , kindly provided by Dr.
B. Posner, Pennsylvania State University. Expression of TR1.9 was
performed as described (13) with some modifications. In brief,
BL21 cells bearing the pTG119 and the pBP101 plasmids were grown at 37
°C in Luria Bertani medium containing 30 µg/ml kanamycin and 10
µg/ml tetracycline (both from Sigma) until the
optical density of the cells reached 0.8 (600 nm). Protein expression
was induced by addition of 1 mM isopropyl-thio-D-1-galactopyranoside (Sigma) for 4 h at 37 °C. Cells were pelleted
and processed as described previously(3) . TR1.9 Fab was
affinity-purified using goat anti-human IgG-coupled Sepharose beads
(ZYMED Laboratories, South San Francisco, CA). After elution with
glycine buffer, pH 2.5, samples were immediately neutralized with 1 M Tris, pH 7.4, and the buffer was then exchanged to 10 mM Tris, pH 7.4.Crystallization
Crystals of TR1.9 Fab were
obtained by a sparse matrix screening procedure (14) using
Crystal Screen (Hampton Research, Laguna Hills, CA). Crystals grew from
drops suspended over wells containing 1 ml of 8% (w/v) polyethylene
glycol 8000, 0.1 M Tris-HCl, pH 8.5, with the drops containing
2 µl of protein (10 mg/ml in 10 mM HEPES, pH 7.0, 0.02%
sodium azide) and 2 µl of well solution. The crystals exhibit the
symmetry of space group P2, with cell constants: a = 50.93, b = 62.78, c =
84.79 Å, and 
= 107.2 degrees.Crystallographic Data Collection and
Processing
X-ray intensity data were collected on an R-AXIS II
system using graphite-monochromatized CuK
radiation from a Rigaku
RU200 rotating-anode generator. All the data used in the analysis were
obtained from one crystal of approximate size, 0.2 
0.2
0.3-mm
, at room temperature. The data were processed using
the program XDS(15) . Statistics for the intensity data are
given in Table 1. Data completeness at various resolutions and F/
(F) levels are presented in Fig. 1.
The average redundancy of the data was 3.5 and the R
on intensities was 8.4%.
1, 2, 3, 4, 5, and 6
(F).
Crystal Structure Determination
The crystal
structure was determined by Molecular Replacement using the program
AMoRe(16) . The Fab fragment of antibody 3D6 (17) (available from the Protein Data Bank (18, 19) as Entry 1DFB), the only human Fab of known
three-dimensional structure with a light chain, was used as the
probe, with separate searches for the V
:V
and
C:C1 modules. The 95% most intense reflections
in the resolution range 8-4 Å were used in the analysis.
The results of the rotation and translation searches are summarized in Table 2. Since space group P2 has an undefined origin
along y, a relative translation search was performed between
the two probe modules. After rigid-body refinement, the correlation
coefficient between calculated and observed structure factors was 0.465
and the crystallographic residual, the R-value, was 40.6%. The
packing of the molecules in the crystal was very reasonable. The
-carbons at the end of V and at the beginning of
C were 7.2 Å apart, while those at the end of V and at the beginning of C1 were 7.3 Å apart,
demonstrating that Molecular Replacement had positioned the two modules
properly relative to each other. As a test, a composite model including
all four domains was used as the probe in another search with AMoRe.
The results (see Table 2) were again unambiguous, with a
correlation coefficient of 0.448 and an R-value of 40.7%.
There is one Fab in the asymmetric unit.
Structure Refinement
All subsequent refinement was
done using X-PLOR(20) . Another rigid-body refinement, using
data for which F 3 (F) in the
10-4.0-Å resolution range and allowing the four domains of
the Fab to move independently of each other yielded an R-value
of 39.0%. At this point, 3D6 residues were replaced with alanines at
the 20 positions in V and the 67 in V where 3D6
and TR1.9 differ in sequence (or with glycines where TR1.9 has this
residue) (Table 3). In addition, the 3D6 residues 91-96 (in
CDR3-L) in the light chain and residues 96-100 (in CDR3-H) in the
heavy chain (following the numbering convention of Kabat et
al.(21) ) were excised, since 3D6 and TR1.9 differ in the
number of amino acids in these two CDRs. The valine at position 225 in
C1 was replaced by the germline alanine (Kabat et
al.(21) . The R-value was 41.6% for this mutated
molecule with data in the resolution range 10-2.2 Å with F 3 (F) (16,475 reflections).
F
map was computed
and displayed with the mutated molecule using the graphics program
FRODO (22) . The fit of model to map was very good. There was
density for most of the omitted side chains and for the excised CDR3-L
and CDR3-H segments (Fig. 2). The omitted side chains were
manually built into the structure on the basis of the map, as well as
the excised regions. Adjustments were made in the NH
and
COOH termini, in the switch regions, as well as in several loops. After
the first rebuilding, the R-value was reduced to 37.4%. A
second round of model rebuilding based on a 2F
- F map further reduced the R-value to 34.8%. A third round of rebuilding only reduced the R-value to 34.6% and manual rebuilding was disconti
- F
map of TR1.9 Fab after rigid-body refinement, with the
CDR3-L loop from the final model overlaid. Although residues
91-96 had not been included in the structure factor calculation,
the map has continuous electron density corresponding to those
residues. The contour level is 1.0 .
2 (F) in the resolution
range 10-2.2 Å (17,852 reflections). One run of simulated
annealing reduced the R-value to 25.3% and four cycles of
alternating thermal factor (B-factor) and positional refinement further
reduced the R-value to 20.7%. The R-value for the
2,422 reflections between 2.2- and 2.0-Å spacings and which had
not been included in the refinement up to this point was 30.7%. - F and F - F maps. The maps
clearly showed that the residue at heavy chain position 225 is not an
alanine, as had been assumed on the basis of the germline sequence. The
electron density was consistent with a valine, as in antibody 3D6, and
the appropriate change was made. Furthermore, putative solvent (water)
molecules were identified. 2
(F) (20,274 reflections), were included in the
refinement (R-value = 23.5%). Three more cycles of
alternating B-factor and positional refinement reduced the R-value to 18.0%.-carbons, 31.1
(r.m.s.d. = 16.6) for the 1756 main chain atoms, and 31.7
(r.m.s.d. = 17.2) for all atoms in the protein. Five solvent
molecules, for which the B-factor was greater than the average for main
chain atoms plus 3 standard deviations (80.9 Å squared), were
discarded. Four protein segments have B-factors greater than this
value: the last three residues in both light and heavy chains, the
first two residues in the heavy chain, and the segment 135-142 in
the heavy chain. These segments are represented by very weak electron
density and could not be positioned with confidence. A plot of the
B-factors is presented in Fig. 3.
values which lie just outside
allowed regions(23) : Ser
and Ala
in
the light chain and Val and Thr
in the heavy
chain (numbering convention of Kabat et al.(21) ). The
average error in atomic positions, calculated according to the method
of Luzzati(24) , is 0.26 Å. Atomic coordinates and
structure factors have been deposited in the Protein Data Bank (entry
codes 1VGE and R1VGESF).
Description of the Structure
A ribbon diagram of
TR1.9 Fab is presented in Fig. 4. As in other Fabs, the
homologous domains of TR1.9 Fab are related by pseudodyads: 174.0
degrees between V and V, and 168.5 degrees
between C and C1. The elbow bend of TR1.9 Fab
is 134.1 degrees. These values are within the range observed for other
Fabs (see Refs. 12, 25, and 26) and, in fact, are very nearly the same
as those observed for Hil, ()a human IgG, 
myeloma protein (PDB entry 8FAB, second Fab in the entry).
Comparison with Other Human Antibody Structures
A
comparison of the individual domains of TR1.9 Fab with those from other
human immunoglobulins is presented in Table 4and Table 5and in Fig. 5and Fig. 6. Atomic coordinates
for five human V domains are available from the Protein Data Bank:
from antibody 3D6 (PDB entry 1DFB), from immunoglobulin Pot (30) (PDB entry 1IGM), and from the Bence-Jones proteins Rei (31) (PDB entry 1REI), Wat (32) (PDB entry 1WTL), and
Len ()(PDB entry 1LVD), and for five human V domains: from antibody 3D6, and from the immunoglobulins Pot, New (33) (PDB entry 7FAB), Kol (34) (PDB entry 2FB4), Hil,
and Mcg (35) (PDB entry 1MCO). Atomic coordinates for only one
C domain, that of the antibody 3D6, is available from the Protein
Data Bank. Coordinates for five C
1 domains are available: those
from antibody 3D6, and those from the immunoglobulins Kol, New, Hil,
and Mcg. The V and V comparisons were made on the
basis of the 72-residue positions which have been found to be
structurally equivalent in V and V domains,
while the C and C1 comparisons were made on the basis of 63
equivalent positions (12) .
-carbon trace of
the V domain of TR1.9 (top) and superposed on the
other human V domains of known structure (bottom).
-carbon trace of
the V domain of TR1.9 (top) and superposed on the
other human V domains of known structure (bottom).
is found to be
very similar in three-dimensional structure to the other human V
domains (Table 4, Fig. 5). Indeed, all the human V
domains are seen to be very similar to each other and, with the
exception of the CDRs, are essentially superimposable. The average
difference among these V domains is 0.49 Å (S.D.
= 0.01); TR1.9 V differs from the other human V
domains on average by 0.42 Å (S.D. = 0.03). These numbers
are essentially the same as those obtained when various structures for
hen egg white lysozyme, crystallized in different space groups and
independently analyzed, are compared (average difference for C
positions is 0.41 Å (S.D. = 0.02) (for PDB Entries 1HEL
(tetragonal) (36) , 132L (orthorhombic)(37) , and 1LYS
(monoclinic, with two molecules per asymmetric unit)(38) ). of TR1.9 differs from that of 3D6 on average by
0.34 Å, again showing a close similarity, although not
unexpectedly since the 3D6 C domain was used as the search
probe in the Molecular Replacement analysis as well as the initial
model for the refinement of the TR1.9 C domain. domains (Table 5, Fig. 6). Excluding Mcg (see below), the average
difference among the human V domains is 0.63 Å (S.D.
= 0.05); TR1.9 V differs from the other human
V domains on average by 0.72 Å (S.D. = 0.03).
As shown in Fig. 6, there are large differences in the
structures of the CDR loops, especially in CDR3-H. In addition to the
variation in the CDRs, there are differences in the
NH-terminal segment (especially with TR1.9 which has an
extra residue inserted after the fourth position) and in other loop
regions.1 domains is
presented in Table 5. Again excluding Mcg (see below), the
C1 domains are seen to be very similar, the average
difference being 0.42 Å (S.D. = 0.01); the TR1.9
C1 domain differs from those of the other human antibodies
on average by 0.39 Å (S.D. = 0.04). but also in the C1 (Table 5). The
consistently larger differences found in the comparisons involving the
Mcg domains probably reflect the low resolution of the Mcg structure
(3.2 Å). Most of the other structures had been determined at
relatively high resolution: TR1.9 Fab at 2.0 Å, New Fab also at
2.0, Kol Fab at 1.9, Hil Fab at 1.8, Rei V at 2.0, Wat
V at 1.9, and Len V at 1.8, although the Pot Fv
structure was determined at 2.3-Å resolution and 3D6 Fab at only
2.7.The Somatic Mutations in TR1.9
The V region of the TR1.9 heavy chain appears to be derived by somatic
mutation from the germline gene V1-3b(39) , also known as
DP-25(40) . The V region of its light chain is most
closely related to the germline gene A`(21) , also known as
L4/L18(41) . An alignment of the TR1.9 sequences with the
closest germlines is shown in Table 6.
termini which are primer-derived,
there are 15 mutations which appear to have occurred in the light and
heavy chains of TR1.9 relative to germline. We are unable to relate the
CDR3-H segment to any of the known D (diversity) segments. The joining
segment for the light chain variable domain is J4 and that for the
heavy chain is J4(21) . and 10 in V; six of these are in CDRs (Table 6). All five changes in V involve residues
that have outward-pointing side chains; four are accessible to solvent
(Asn
, Ala
, Arg
, and Asn
in CDR2-L), while the fifth is partly buried (Ile
).
Of the 10 changes in TR1.9 V, two are buried in the domain
interior (Leu
in CDR1-H and Phe
); six of the
eight non-glycine residues have side chains that are outward-pointing:
four are exposed to solvent (Ser
, Thr
, and
Arg
in CDR2-H, and Pro), while two are partly
buried (Ser
in CDR2-H and Thr
). None of the
putative somatic changes occurs at a position that is involved in the
V:V interaction (Table 6). The putative
somatic mutations which appear to have occurred in TR1.9 are portrayed
in the three-dimensional structure of the molecule in Fig. 7.
is on the left (lighter shading) and V is on the
right (darker shading). The residues which differ from germline are
indicated by filled circles; those in the CDRs are drawn
larger. The residues in CDR3-H are indicated by empty circles.
The NH and COOH termini of both chains are
labeled.
-terminal
segment of TR1.9 V is the result of the use of a 1a/3a
oligonucleotide primer for amplification(5) . The insertion of
this extra residue causes a structural rearrangement in this part of
TR1.9 V (relative to the other known V structures) (Fig. 6). The fact that TR1.9 still displays
high affinity for TPO strongly suggests that the NH terminus of V is not involved in the interaction with
the antigen.Conclusions
In the absence of a three-dimensional
structure for the complex of TR1.9 with TPO, we can only guess at the
structural basis for the high affinity of the binding. The 7-8
kcal/mol required to increase the affinity from weak (say, K
10
-10
M) to strong (e.g. K 10M) binding could be derived
from the formation of salt bridges, or of hydrogen bonds, especially
when involving charged groups(42) . Comparing TR1.9 to the most
closely related germlines (Table 6) reveals that replacements
involving charged residues appear not to have occurred. However, the D
segment and the J used to construct the heavy chain
variable domain of TR1.9 produced four charged residues in CDR3-H: two
aspartic acids, one glutamic acid, and one lysine. These charged
residues, if they form salt bridges with oppositely charged residues in
TPO, may be responsible, in part or in whole, for the high affinity of
the interaction. are mutated to serine
and threonine, respectively, in TR1.9. Ser-H in TR1.9 is
at the start of the loop structure in CDR2-H and Thr-H is
in this loop. The murine antibody 36-71 (44) (PDB entry
6FAB) and the humanized murine antibody H52 (45) (PDB entry
1FGV) have asparagines at both positions. In antibody 36-71, the side
chain of Asn-H forms a hydrogen bond with the main chain
while Asn-H does not; in antibody H52, both asparagines
form hydrogen bonds with the main chain. The replacement of the
germline Asn-H and Asn-H should result in a
reduced stability and greater flexibility of this part of CDR2-H,
especially since two glycines are present in this segment. Another part
of TR1.9 that is almost certainly flexible is the CDR3-H loop which
features three glycine residues in a row. The CDR2-H and CDR3-H loops
abut each other and together occupy a central position in the combining
site (Fig. 7). Many residues in the CDR2-H and CDR3-H loops are
often found to be involved in ligand binding in other
antibodies(12, 28, 46) . It is tempting to
speculate that increased flexibility and deformability, (
)made possible by the presence of the glycines and the
reduced number of asparagines, improve the binding of TR1.9 to TPO, in
the manner of an ``induced fit'' (47) .
))))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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