(Received for publication, April 26, 1995; and in revised form, July 26, 1995)
From the
Comparison of seven high resolution x-ray structures shows that
the conformations of canonical complementarity determining region (CDR)
loops, which are shared by these antibodies, are very similar. However,
large spatial displacements (up to 2.7 Å) of the essentially
identical CDR loops become evident when the antibody -sheet
frameworks, to which the loops are attached, are least-squares
superposed. The loop displacements follow, and amplify, small
positional differences in framework/loop splice points. Intradomain
structural variability and, to a lesser extent, domain-domain
orientation appear to cause the observed loop divergences. The results
suggest that the selection of framework regions for loop grafting
procedures is more critical than previously thought.
Immunoglobulin variable domains, VL ( We
have compared, via least-squares superpositions, the Fvs of seven x-ray
structures refined to high resolution (better than 2 Å) available
in the Brookhaven Protein Data Bank(6) . It was anticipated
that a systematic comparison of structures determined to such high
precision would (i) shed light on the general relation between CDR loop
conformation and position and (ii) help to assess the limitations of a
procedure widely used in comparative model building, i.e. splicing of loops from a known x-ray structure onto the conserved
structural scaffold of an antibody model(7, 8) . Fig. 1shows a comparison (9) of the amino acid
sequences of the seven antibodies, which include various heavy and
light chains (both
Figure 1:
Sequence profile of antibody light (a) and heavy (b) chain variable regions. The Kabat
numbering scheme (3) is used with the exception of the L1 and
H1 regions where it is modified according to (5) .
Hypervariable regions (3) and CDR loops (4, 5) are boxed in magenta and white, respectively. Structural determinants for each
canonical CDR loop are labeled (above the residue) with the respective
CDR designation (L1, L2, L3, H1, H2). Residues shared by superposition
sets S1 and S2 are shown on a green background. Residues
unique to S2 are shown on a red background. CDR loops compared
in Fig. 2and Table 1and Table 2are shown on an orange background.
Figure 2:
Comparison of canonical CDR loops.
Backbone (N, C
Two different measures
of differences in loop conformations were calculated. The ``direct
rms'' value (d_rms) was calculated for the direct, pairwise,
least-squares superposition of the CDR backbones only. This value can
be regarded as a similarity measure of the loop conformations per
se, regardless of any structural context. The ``spatial
rms'' (s_rms) value, on the other hand, was calculated for the
loop backbones overlaid as a result of the superposition of the
conserved framework regions, i.e. the S1 and S2 sets. Thus,
the s_rms value includes not only the differences in CDR loop
conformations but also those in CDR loop positions. This value reflects
the take-off angles and the ensuing rigid-body shifts in the spatial
orientations of CDR loops. A comparison of two CDR loops, which have a
similar conformation but are spatially displaced, will yield a low
d_rms and a high s_rms value. Analysis of loop conformations shows
that all seven antibodies in our comparison set share canonical
structure types (5) for CDR loops H1 and L2 and that three
antibodies share equivalent canonical structures for CDR loops H2 and
L3. Table 2lists the results of the pairwise rms comparisons for
the H1, H2, L2, and L3 loops with equivalent canonical structure. As
can be seen, the backbone conformations (d_rms) of loops from the same
canonical group are remarkably similar ( Relative orientations of the VH-VL domains in the Fvs
included in our comparison showed variations typically seen
before(12) , i.e. up to In
addition to the s_rms deviations reported in Table 2, we have
also measured s_rms values for CDRs after independent superposition of
the isolated VH and VL domains, respectively. These calculations were
carried out for VH and VL domains that share equivalent canonical
structures for H1 and H2 and for L2 and L3, respectively. The results
are shown in Table 3. The comparison shows that the VH-VL domain
orientation contributes to the positional differences of the CDRs.
However, large s_rms values are also observed after superposition of
single domains. In this comparison, intradomain framework/loop
variability appears to be the primary cause of loop positional
differences in both VH and VL domains, contributing, on the average,
about 75% to the structural variability (as expressed in terms of s_rms
values). Therefore, effects such as differences in side chain packing
of the
Fig. 3shows the superposition of the Fv framework regions and
illustrates the presence of systematic structural deviations. The
central strands of the
Figure 3:
Comparison of framework regions and end
points. The S1 set superposition of the framework
The
analysis has implications for comparative modeling of an unknown
antibody binding site (i.e. the Fv), which usually proceeds in
two steps: (i) a framework is chosen from among the available x-ray
structures via amino acid sequence alignments and (ii) desired CDR
loops are identified and implanted onto the selected
framework(7, 8) . Given the many x-ray
crystallographic structures of antibody fragments now available, the
framework selection has generally been considered as a straightforward
and almost trivial exercise. However, the data presented here suggest
that the subtle positional differences in framework end points define
an inherent limit to the accuracy of CDR loop modeling even if
high-resolution structures are used as templates. Although the
canonical loop motifs in general agree to within 0.5-Å d_rms,
framework-dependent differences in loop take-off angles set the actual
accuracy limit, i.e. the s_rms value, to at least 1.5 Å
per loop. For example, the error would be 1.9-2.4 Å if the
J539 H2 loop were implanted onto the HIL framework after Fv
superposition. Even if it were possible to accurately model VH-VL
domain orientation angles, relative spatial displacement of CDR loops
would not be controlled. The choice of framework regions and the
definition of loop/framework junctions are important components of
model-building protocols employing loop transfers and loop simulations.
The exact position of the splice point or framework terminus will
strongly influence the take-off angle of the loop and, in consequence,
its spatial position. The humanization of antibodies presents a
similar problem. In this case, the rodent CDRs are experimentally
grafted onto human framework regions(13) . Despite conservation
of murine structural determinants, moderate loss of antibody affinity
is often observed(14) . This may arise from changes in CDR loop
position relative to the framework and to each other and may explain
why the selection of a highly homologous human framework template is
often critical(15) . In summary, our comparison of high
precision x-ray structures confirms the similarity of equivalent
canonical conformations and shows the presence of significant
systematic differences in their spatial positions. The analysis implies
that procedures for the selection of the best possible framework
template in antibody modeling and humanization should be given much
more attention than in the past. Future assessments of prediction
accuracy should be expressed in, and discussed with respect to, the
s_rms similarity measures in addition to the more commonly used, and
better looking, d_rms values.
)and VH,
associate noncovalently to form the Fv, a dimer of antiparallel,
eight-stranded
-sandwiches(1, 2) . The VL and VH
-sheets from different antibodies are nearly identical in three
dimensions. However, the six complementarity determining region (CDR)
loops (L1-L3, H1-H3), which connect the
-strands of
the conserved framework and encode antigenic specificities, are much
more variable in both sequence and conformation (2, 3) . Chothia, Lesk, and colleagues (4, 5) identified sets of similar
``canonical'' conformations for all CDR loops except H3.
Between 50 and 95% of antibody sequences are consistent with the
classified canonical conformations(4) , which are determined by
conserved interactions of only a few key residues (``structural
determinants'') within the loop and/or the framework regions. Some
differences in the position of canonical CDR loops relative to
superposed framework regions by comparing x-ray structures (4) or x-ray and modeled structures (5) were previously
observed. However, these effects were observed in structures determined
at medium resolution, generally considered
minor(4, 5) , and not systematically explored.
and
) from free as well as
antigen-complexed antibodies. The 4-4-20 Fv, the highest resolution
structure with
light chain, was used as the template on which
backbone segments of the other Fvs were superposed. Cumulative backbone
root mean square (rms) deviations of the
-strands were determined
after superposing each of these antibodies on the 4-4-20 Fv. Two
alternative least-squares superpositions were used, employing different
pairs of equivalence residues. The S1 set of residues consisted of only
the most conserved regions of the Fvs, i.e. the four short
4-residue segments (10) of the central
-sheets. The S2
set consisted of a more extended set of residues and included the
majority of the
-sheet framework (Fig. 1) akin to Stanfield et al.(11) . As can be seen in Table 1, the
cumulative backbone rms deviations were small and the results obtained
with the two superposition sets were similar.
, C, O) comparisons of equivalent CDR loop
structures are shown: a, H1; b, L2; c, H2.
The top of each figure shows the direct least-squares fit of
the loops (corresponding to d_rms values); the bottom shows
comparison after S1 set framework superposition (corresponding to
s_rms1 values). Color code: 4-4-20, magenta; D1.3, green; H52, yellow; HIL, red; J539, silver; KOL, blue; S.E.155-4, gold.
0.5 Å for H1 and
0.2 Å for L2) in the high resolution structures. In
contrast, the s_rms values show much greater differences. The
superposition sets S1 and S2 gave similar cumulative backbone rms
deviations for the framework segments (d_rms in Table 1) and
similar s_rms values (s_rms1 and s_rms2, respectively) for the compared
CDR loops. Fig. 2provides graphical demonstrations of the CDR
loop d_rms and s_rms superpositions. The results indicate that
positional differences of CDR loops are a rule in highly refined
antibody x-ray structures. Thus, whereas the conformations of
equivalent canonical CDR loops are very similar in the set of highly
refined antibody x-ray structures, their spatial positions are
different.
10°. It should be
noted that our way of superposing and comparing the Fv structures
differs fundamentally from conventional comparisons, which rely on
optimal least-squares superposition of one domain followed by
measurement of angular deviation (from the pseudo-dyad axis of the Fv)
of the other domain. Since different variable domains have different
primary structures, numbers of residues, and molecular masses, the
results of superpositions of selected domain pairs are expected to vary
somewhat from case to case, which is critical for Fv comparison. Thus,
we have tried to employ a more uniform procedure to compare Fvs by
superposing the few invariant VL and VH residues that participate in
the formation of the VH-VL domain interface(10) .
-sheet framework, CDR-CDR interactions, or intermolecular
interactions in the crystal environment must contribute to positional
CDR loop differences. These spatial deviations cannot simply be a
consequence of antigen binding as they are observed even when comparing
the five structures crystallized in the absence of antigen.
-sheets gradually diverge toward their
termini, and edge strands often display even wider overall differences.
The positions of the framework termini differ with average pairwise
differences of the terminal
-carbon positions of typically
1
Å or more. Relative displacements of CDR loops roughly correlate
with positional differences at the CDR-framework junctions. By
comparing some Fvs, we noted concerted displacements of light and heavy
chain CDR loops, suggesting a role of CDR-CDR interactions.
-strands
(
-carbon traces) that are connected by CDR loops (C/B, CDR 1; C`/C", CDR 2; F/G, CDR 3) of the variable light (yellow labels) and heavy chains (lightblue labels) is
shown, color coded as in Fig. 2. Except for CDR H3, framework
end points (highlighted in white) are in accordance with
canonical loop definitions(5) . The view is facing down the
antigen binding sites. The complete backbone of the 4-4-20 Fv is traced
in magenta.