(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, 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.
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), ()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) .
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 1, 2, 3, 4, 5, and 6
(F).
A
2FF
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
nued for the time being.
Figure 2:
Stereodrawing of a portion of the
2F - 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
.
The structure was then refined using X-PLOR with
data for which F 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%.
Another round of model rebuilding using FRODO was performed based on
2F - 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.
From this point onward, all the data in
the resolution range 10-2.0 Å, for which F 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%.
The average B-factor is 30.9 Å
squared (r.m.s.d. = 16.7) for the 439 -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.
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 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).
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 -carbon trace of
the V
domain of TR1.9 (top) and superposed on the
other human V
domains of known structure (bottom).
Figure 6:
Stereodrawing of the -carbon trace of
the V
domain of TR1.9 (top) and superposed on the
other human V
domains of known structure (bottom).
The TR1.9 V 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) ).
The C 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.
A
greater variation is observed for the human V 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.
The comparison of the human C1 domains is
presented in Table 5. Again excluding Mcg (see below), the
C
1 domains are seen to be very similar, the average
difference being 0.42 Å (S.D. = 0.01); the TR1.9
C
1 domain differs from those of the other human antibodies
on average by 0.39 Å (S.D. = 0.04).
In this collection,
the immunoglobulin Mcg is found to be the most different, not only in
V but also in the C
1 (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.
Ignoring the
differences at the NH 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 J
4 and that for the
heavy chain is J
4(21) .
Relative to the closest
germline, five somatic mutations appear to have occurred in TR1.9
V 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.
Figure 7:
Ribbon drawings of the Fv of TR1.9 viewed
from the side (top) and end-on (bottom). V 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.
The insertion of an extra residue in the NH-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.
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 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) .
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.