From the
Antibody gene sequences, particularly those of
Antibody combining sites, arising by a combinatorial
recombination of a limited number of gene segments, can be
complementary to almost any antigenic structure. The basic shape of the
variable domains V
This knowledge is based on about 30 crystal structures of
antibody variable domains and about 10
In this study, we report a mouse
On-line formulae not verified for accuracy The total concentrations of antibody and soluble antigen are given
by Ab
V
For a
model of the insertion, we assumed that it would form a
The antibody 93-6, directed against the
While the heavy chain is a typical member of mouse heavy chain
subgroup V(a) (Fig. 1A), the
To
determine whether these deviations from the consensus
In an antigen
binding assay of E. coli periplasmic extracts, only the wild
type protein and the EL
A more extensive repacking is supported by the fact that
two other highly conserved residues of mouse
An attempt was made to find the correct docking
orientation of 93-6 to tryptophan synthase by analyzing the
electrostatic potential of the two molecules in the DELPHI module of
the INSIGHT II modeling package. This approach was not successful as
both molecules showed a patchy distribution of positive and negative
potentials without clear complementarity. Nevertheless, an approximate
docking orientation can be derived from steric considerations: almost
all orientations other than an almost perpendicular orientation of the
V
It is currently unclear by which mechanism the two deviations from
the consensus
In conclusion, this functional antibody shows
that conservation of consensus structure is not necessary for function.
On the contrary, conversion to the consensus in FR2 makes the antibody
unable to bind its antigen. We suggest therefore that even those CDRs
which are restricted in length, as well as adjacent stretches of
framework residues, should be included in antibody mutagenesis schemes,
as the consensus framework may provide only a subset of potentially
good binders.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. Michel Goldberg (Institut Pasteur) for
the hybridoma 93-6, purified
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
light
chains, are very well conserved in the framework region, and the
variability is concentrated in the complementarity-determining regions
(CDR). We now found that the murine antibody 93-6 (Djavadi-Ohaniance,
L., Friguet, B., and Goldberg, M.(1984) Biochemistry 23,
97-104) whose F
fragment binds the
-subunit of
Escherichia coli tryptophan synthase with high affinity
(K
of 6.7
10
M) has a highly unusual
light chain framework,
which is crucial for the function of this antibody. It carries an
insertion of 8 amino acids in a conserved framework loop that faces the
antigen, and its framework region 2 (FR2) which precedes CDR2 is
shortened by one amino acid, normally leucine and part of an absolutely
conserved
-bulge preceding CDR2. Removal of the insertion to
restore the consensus sequence reduced the binding affinity of 93-6 by
a factor 3, while insertion of the missing leucine into FR2 completely
abolished binding.
and V
is a
-sheet
sandwich, and the folding pattern of the strands is termed the Greek
key motif. The variability is concentrated in six surface-exposed
loops, the complementarity-determining regions (CDRs).
(
)
Length variations in these loops are important for the
shape of the combining site. The length diversity of the first two CDRs
is given by the diversity of the V genes. In contrast, the length
variation of the CDR3 of the heavy chain involves the junctional
diversity of various V, D, and J segments as well as noncoded bases.
The framework regions are highly conserved between and within variable
domain classes and essentially no insertions and deletions are
observed.
sequences, mostly of
human and murine origin (Kabat et al., 1991; Rees et
al., 1994). For man, probably all V
genes (Chothia
et al., 1992; Tomlinson et al., 1992), most
V
genes (Cox et al., 1994; Weichhold et
al., 1993; Lautner-Rieske et al., 1993) and many
V
genes (Williams and Winter, 1993) are known. In
mouse, in contrast, our knowledge is based mostly on sequencing of many
individual monoclonal antibodies. It is thus currently unclear what
percentage of mouse V
and V
genes are
known. In mouse, there are only three V
genes (Selsing
et al., 1982; Sanchez et al., 1990), and they are
rarely used. From the known sequences, and especially from the known
structures, a picture emerges with V
genes particularly
well conserved (Kabat et al., 1991; Rees et al.,
1994), limiting the structural variations to the CDR regions and only
displaying somatic point mutations in a series of very similar
frameworks.
chain, found in a
functional antibody, which is different from any other in the data base
in two absolutely conserved features. We also show that it loses
function when converted back to the consensus.
Cloning
Hybridoma 93-6 (IgG1) (Djavadi-Ohaniance
et al., 1994) was grown in RPMI 1640 medium, supplemented with
heat-inactivated horse serum, pyruvate, penicillin, and streptomycin.
270 µg of mRNA were isolated from 5 10
cells
using the mRNA isolation kit from Pharmacia Biotech Inc. 6.7 µg of
mRNA were used as template for cDNA synthesis using the Pharmacia 1st
strand cDNA synthesis kit. NotI-d(T)
or 3`
primers (specific for C
1 and C
) were used as
primers. The PCR, carried out as described by Huse et
al.(1989) using cDNA mRNA hybrids as the template, gave rise to
fragments of the expected sizes. V
C
1 and
V
C
genes were cloned into the vector pSL301
(Invitrogen Corp.) as XhoI-SpeI and
HindIII-SacI fragments and sequenced. In a separate
experiment, the primer sets for V
and V
from
the antibody PCR kit from Pharmacia were used instead, with subsequent
assembly to a scFv as described by the manufacturer. The same sequence
was obtained as with the Fab primers modified from Huse et
al.(1989).
Expression of the scFv Fragment
The scFv fragment
of the antibody 93-6 was assembled by PCR of the Fab fragment and
subcloned into the pIG6 expression vector using primers SC-1, -2, -3,
and -4 (Ge et al., 1995). The extra loop (EL) in FR3 was
converted to the consensus sequence (QNRSPFGNQLN GTD) by
site-directed mutagenesis using a Bio-Rad kit, whereby a BspEI
site was destroyed. The ``missing'' leucine (Leu-47) was
added (L
, EL
L
) by
site-directed mutagenesis with an oligonucleotide containing a
HindIII site. The wild type as well as the mutants
(EL
, L
,
EL
L
) were confirmed by DNA
sequencing. The clones of pIG6-scFv(93-6) wild type,
EL
, L
, and
EL
L
in Escherichia coli JM83 were grown in LB medium containing streptomycin (25
µg/ml) and ampicillin (100 µg/ml). The fresh cultures of
OD
0.4-0.6 were induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside and shaken at room
temperature for 3 h. The periplasmic fractions were obtained and scFv
fragments were purified by immobilized metal ion chromatography as
described elsewhere (Ge et al., 1995; Lindner et al.,
1992).
Cloning and Expression of Tryptophan Synthase
The tryptophan synthase -Subunit
-subunit was PCR
amplified from the genome of E. coli with one primer encoding
an N-terminal addition of 6 histidines. This is structurally far
removed from the known epitope for the antibody 93-6 (Friguet et
al., 1989, 1993). The protein was purified by immobilized metal
ion chromatography on Ni-nitrilotriacetic acid (Lindner et
al., 1992).
ELISA
The antigen was coated at 2 µg/ml on
special luminescence ELISA plates (Dynatech), and the wells were
blocked with milk powder. The scFv fragments were detected with the
anti-myc-tag antibody 9E10 (Munro and Pelham, 1986) and a goat
anti-mouse alkaline phosphatase conjugate. The phosphatase activity was
detected with the ELISA-light kit using disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2`-(5`-chloro)tricyclo[3.3.1.1]decan}-4-yl)-phenyl
phosphate (Tropix) in a ML3000 luminometer (Dynatech).
Determination of Binding Constants
In the
measurements of the binding constants, 310
M to 10
M scFv fragment was
incubated with varying amounts of tryptophan synthase
-subunit in
0.9 ml of phosphate-buffered saline containing 1 mg/ml bovine serum
albumin at 4 °C for 16 h. Aliquots of 0.2 ml were removed for
determining the unbound scFv, and the amount of tryptophan synthase
used in coating was first tested to be small enough to not displace the
equilibrium (Friguet et al., 1985). Triplicate measurements on
two different protein preparations were used for each mutant. The
binding constant was obtained by fitting the ELISA signal directly to
the law of mass action using the relation A =
A
(1 -
[Ab
Ag]/[Ab
]),
where A and A
are the measured absorbance
and the value in the absence of soluble antigen, respectively, and
and Ag
,
respectively, and K
is the dissociation
constant.
Modeling
Of the V sequences in the
Protein Data Bank (PDB; Brookhaven National Laboratories, April 1993),
those of antibodies 36-71, R19.9, and HyHel5 were most similar to
V
of 93-6 (73-77% identity). The similarity to other
antibodies was significantly lower. The 93-6 V
main chain
was modeled on R19.9, except for CDR H2, which was modeled on HyHel5
because this antibody also binds a protein rather than a small ligand.
The model of CDR H2 must be considered with caution, since it contains
three consecutive glycines that are largely unconstrained by the rest
of the molecule. CDR H3 of 93-6 was shorter than that of 19.9 but also
contained residues Arg-94 and Asp-101 that form a salt bridge and have
a conserved structure in all antibodies that contain them. Only four
residues were left in 93-6 to close the loop between Arg-94 and
Asp-101, and because the second of these was a glycine, it was
considered likely that the loop would form a type II` turn (Sibanda
et al., 1989). As a control, CDR H3 was modeled on CDR H3 of
AN02, the only one in PDB to have the same length. The cis-Pro
in AN02 was excised, and the backbone was converted to a trans conformation, then steric clashes were relieved manually and the
loop was energy-minimized in vacuo without charges and with a
planarity constraint on the peptide bonds. The final structure was very
similar (main chain root mean square deviation <1 Å) to the
one obtained in the other approach. After modeling the main chain of
V
, the side chains were modeled by maximum overlap to
36-71, R19.9, and HyHel5. In cases of discrepancy between the
structures, which were few and generally seen in solvent-exposed
positions, the structure with the most favorable torsion angles (Ponder
and Richards, 1987) was used. The side chains in loop H3 were added
after building the V
-V
dimer.
was most similar to a mutant of McPC603 containing the CDR1
region of MOPC167 (1IMN) (55% identity) and also had CDR loops of the
same length, so that this protein was used as a template. For CDR L1, a
certain degree of uncertainty stems from the fact that, although all
four CDR L1 loops of this length in PDB (B13I2, 4-4-20, 26-10 and
McPC603/MOPC167) form type I + G1-bulge turns, their position
relative to the light chain framework is fairly variable and all four
have Gly at position 29, while 93-6 has Ser at this position and Gly at
position 28. To explore alternate conformations, we extracted all 3:5,
5:5, 5:7, and 7:7 loops from the compilation of Sibanda et
al.(1989) that contained Gly in the same position as 93-6. Most of
these were further reclined than CDR L1 of McPC603/MOPC167; the loop
formed by residues 17-23 of staphylococcal nuclease could be
overlapped best with the base of CDR L1 and was included into the model
as an alternative. As described in the text, two models were generated
for the CDR L2 region: one in which the
-bulge was preserved and
the following
-turn was changed to a
-turn, and one where the
-bulge was changed to a
-strand and the framework loop
following CDR L2 was shifted to compensate for the residue missing in
the core. Although we made conservative assumptions about this shift,
the actual structure may undergo more significant alterations since two
highly conserved residues in this area are not present in 93-6: Pro-59
is Ser and Gly-64 is Asp (possibly replacing with its side chain a
conserved internal water molecule in the back of CDR L2).
-hairpin
with a type I turn because of Pro in the i + 1 and Gly in the i
+ 4 position (Sibanda et al., 1989). Using the procedure
of Jones and Thirup(1986), we found eight loops with good main chain
overlap. Because of Gly-69E at the base of the turn (Fig. 1), the
loops showed significant variability. The loop formed by residues
28-37 of 3EBX was best compatible with Pro in position i +
1. We positioned the side chains of 93-6 V
by maximum
overlap to McPC603/MOPC167 after fitting in the insertion but prior to
modeling CDR L2. Residues without correspondence were given the most
favorable torsion angles (Ponder and Richards, 1987) that did not
result in steric overlap. After the CDR L2 models were made, both
structures were subjected to 500 rounds of steepest descents
energy-minimization in vacuo without charges and a fixed
backbone to relieve close contacts. Finally, the V
model
was complexed to the V
model by maximum overlap of its main
chain atoms at the V
-V
interface with V
of R19.9. Side chains were then added for CDR H3 and the entire
model was again subjected to 500 rounds of steepest descents energy
minimization in vacuo without charges and with a fixed
backbone.
Figure 1:
A, Sequence of the heavy chain of the
antibody 93-6 and B, sequence of the light chain, aligned to
the consensus sequences of the six classes of mouse chains as
defined by Kabat et al. (1991). Note that in both chains the
first two amino acids are encoded by the PCR primer and the true
residues are therefore unknown. The numbering according to Kabat is
indicated. X indicates a variable residue with no clear
preference, the dash (-) a deletion. The insertion and the
deletion in the framework of 93-6 are
highlighted.
The approximate docking orientation was derived from
steric considerations, since most relative orientations can be excluded
because of significant overlaps. Only an almost perpendicular
orientation of the V-V
axis relative to the
axis appeared to be possible, in the absence of
major structural rearrangements upon complex formation.
-subunit of
E. coli tryptophan synthase, was obtained by hybridoma
technology (Djavadi-Ohaniance et al., 1984). We amplified the
genes of the Fab fragment by PCR of hybridoma mRNA, using primers
hybridizing in the constant domains C
1 and C
and primers hybridizing to the 5` end of the V genes, which were
adapted from the literature (Huse et al., 1989). To exclude
any PCR artefacts, the mRNA was independently amplified again by PCR
with a different set of commercial primers hybridizing to the 5` and 3`
sequences of the V genes (see ``Materials and Methods'')
(Fig. 1). In both cases, the same antibody sequence was obtained
(DNA sequence submitted to EMBL, accession nos. Z48767 and Z48768).
light chain differs
in two important aspects from any other
chain in the data base.
First, it has an insertion of 8 amino acids in FR3 (after residue 69,
Kabat numbering) (Kabat et al., 1991), which forms a turn at
the antigen binding face of the molecule. Second, the sequence is
missing a leucine residue in FR2 that normally gives rise to a
-bulge at the base of CDR2 (Fig. 1B).
sequence
are of functional importance, we expressed the protein as a single
chain Fv (scFv) fragment in E. coli (Glockshuber et
al., 1990; Ge et al., 1995) and constructed three
mutants: one in which the FR3 insertion is removed by converting the
nontypical 11 residue stretch (highlighted in Fig. 1) to the
consensus sequence GTD (``extra loop deleted,'' denoted
EL
), one in which the missing leucine residue is
inserted into FR2 (``Leu insert,'' denoted
L
), and one that contains both mutations (denoted
EL
L
). The scFv fragments all contain
a shortened 3-amino acid long N-terminal FLAG (Knappik and
Plückthun, 1994) and a C-terminal myc-tag (Munro and
Pelham, 1986) for detection, as well as a C-terminal histidine tail for
purification (Ge et al., 1995; Lindner et al., 1992).
Thus, the amount of soluble periplasmic protein could be determined by
Western blotting with two different antibodies, and it was found to be
very similar for all four variants (data not shown).
mutant gave a positive signal
on an ELISA plate coated with tryptophan synthase
-subunit
(Fig. 2A). Similar results were obtained with purified
proteins. The binding constant for the wild type and the EL
version was then determined in solution (Friguet et al.,
1993), using luminescence ELISA as detection method. The binding
constant determined for the wild type and EL
were
different only by a factor 3 (Fig. 2B), suggesting that
the extra loop does not make significant contact to the antigen. The
failure of the two leucine insertions L
and
EL
L
to bind antigen, observed with
both purified protein and crude periplasmic extracts, was more
unexpected, and may indicate a crucial structural change in CDR2,
normally extremely well conserved. While all the mutant scFv fragments
are soluble in the periplasm, some aggregation of the purified protein
was observed upon concentration, but this appears to correlate more
with the presence of the extra loop than the Leu insertion.
Figure 2:
A, periplasmic extracts of the mutants of
93-6 scFv fragments were analyzed by luminescence ELISA with the
relevant antigen tryptophan synthase, and bovine serum albumin was used
as a negative control. The concentration of soluble scFv fragments was
estimated from Western blots (Ge et al., 1994; Knappik and
Plückthun, 1994) and the amounts of periplasmic extracts used in
the ELISA were adjusted to correspond to identical scFv concentrations.
The height of the bars is given in arbitrary light units. B,
determination of the binding constant of the scFv fragments of the
antibody 93-6 (wild type) () and the EL
mutant
(
) by the method of Friguet et al. (1985). From two
protein preparations, K of the wild type was found to be
5.3
10
M and 1.6
10
M for the EL
mutant. The K of
the non-recombinant F
is 6.7
10
M (Brelier and Goldberg,
1990).
To
obtain a better insight into the potential effects of these mutations
on the structure of 93-6, we built a molecular model for the Fv
fragment (Fig. 3) using standard homology modeling techniques
(see ``Materials and Methods''). As anticipated from the
binding studies, the insertion appears to be too far removed from the
binding pocket to interact efficiently with the antigen. Its
involvement in binding is further hindered by the screening effect of
the large CDR L1. Nevertheless, in future projects, a combination of a
FR3 insertion with a short CDR L1 may become interesting for
engineering antibodies that bind large flat epitopes. A 4-amino acid
insertion into the FR3 loop of the heavy chain of an anti-nitrophenyl
antibody has also been constructed and found to not influence activity
(Simon and Rajewsky, 1992), and this would not be expected for a
hapten-binding antibody, but such an insertion could again be useful
for a large flat antigen.
Figure 3:
A,
stereo view of a model for the interaction of antibody 93-6 with
fragment F2 of the tryptophan synthase -subunit (1WSY residues
B276-B393). The six CDR loops, the light chain insertion and the
solvent-exposed residues of the F2 epitope mapped by Goldberg and
co-workers (Friguet et al., 1989, 1993) are shown in
bold. B, view of the light chain across the binding
pocket and C, top view of the 93-6 binding pocket. CDR loops
(L1, L2, L3, H1, H2, and
H3) and insertion (INS) are shown in bold.
D (left), CDR L1 as modeled on 1IMN is compared to
CDR L1 of 1IGF (thin line) and residues 17-23 of 1SNC
(staphylococcal nuclease; dotted line); (middle) CDR
L2 with the preceding
-bulge preserved and the
-turn relaxed
into a
-turn is compared to CDR L2 with the
-bulge resolved
into a continuous
-strand and the
-turn preserved (thin
line); (right) the most reclined and the most upright
insertion conformation identified from PDB by the method of Jones and
Thirup (1986). The variability stems from the conformational freedom of
Gly-69E (see Fig. 1) at the base of the
turn.
The deletion at residue 47 lies in a
structurally conserved -bulge that is also found in antibody heavy
chains, and in CD2, CD4, and CD8 (Chothia et al., 1985; Jones
et al., 1992; Wang et al., 1990; Leahy et
al., 1992), despite great sequence divergence in this area and
different lengths of the following loop. The deletion could potentially
be accommodated in two ways: (i) the
-bulge could be maintained by
moving Tyr-49 into the protein interior and relaxing the unusual
-turn found in CDR L2 structures into a more common type II`
-turn or (ii) the
-bulge could be resolved into a normal
-strand, leaving the
-turn in CDR L2 intact but causing a
shift in the following framework loop to compensate for the missing
residue in the core. Both possibilities yielded plausible models
(Fig. 3D), with the second obtaining slightly better
scores in the three-dimensional-one-dimensional validation method of
Lüthy et al.(1992). Neither structural model provides a
clear indication why the insertion of the missing leucine has such a
dramatic effect, but it must be considered that the deletion in 93-6
may have caused far-reaching repacking of the hydrophobic core,
ultimately influencing the conformation of several CDRs, while homology
modeling by its very premise attempts to compensate for all changes
locally.
chains are mutated
in 93-6, and both are located in the same part of the V
structure as the framework deletion, namely in the region
outlined by strands C`, C", and D: Pro-59, which in other
chains
determines the structure of the loop connecting strands C" and D, is
replaced by serine in 93-6, and Gly-64, which in other
chains
faces an internal conserved water molecule, is replaced by aspartate.
The latter residue in particular, which in our model replaces the
internal water with its side chain and assumes its interactions with
the central tryptophan (Trp-35), may instead cause a distortion in
strand D that would reorient the charged side chain to the outside and
allow its solvation.
-V
axis relative to the
axis are unlikely because of significant overlaps. An interaction of
the insertion with tryptophan synthase only appears likely if V
is oriented toward the flat side of the
dimer.
structure came about. Insertions and deletions are
extremely rare in the somatic maturation of antibodies (Chen and
Poljak, 1974; Press and Hogg, 1970; Wang et al., 1973; Cumano
and Rajewski, 1986), and in the the present antibody, two such events
would be required. Since, in contrast to the human sequences, only a
fraction of the mouse
genes are known, it remains a possibility
to be investigated in future work whether an unusual V
gene has
remained undiscovered.
/EMBL Data Bank with accession number(s) Z48767 and
Z48768.
-subunit of tryptophan synthase, and
helpful discussions, Birgit Lindner for help in protein purifications,
and Claus Krebber for cloning and purification of
His
-beta-TrpSynthase and helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.