The Rational Construction of an Antibody Against
Cystatin: Lessons from the Crystal Structure of an
Artificial F
ab
Fragment
WolframSchiweck
1,2
andArneSkerra
2
*
1
Max-Planck-Institut fu
È
r
Biophysik, Heinrich-Hoffmann-
Str. 7, D-60528 Frankfurt am
Main, Germany
2
Institut fu
È
r Biochemie
Technische Hochschule
Petersenstr. 22
D-64287 Darmstadt, Germany
In a protein design study the arti®cial antibody M41 was modelled with
its binding site complementary to the protease inhibitor cystatin, which
was chosen as a structurally well-characterized ``antigen''. The modelling
of M41 took advantage of the crystal structure of the anti-lysozyme anti-
body HyHEL-10 as a structural template. Its combining site was reshaped
by replacing 19 amino acid side-chains in the hypervariable loops. In ad-
dition, ten amino acid residues were substituted in the framework
regions. The crystal structure of the corresponding antibody model
M41, which was produced as an F
ab
fragment in Escherichia coli, was
determined at a resolution of 1.95 A
Ê
. The crystals exhibited symmetry of
the space group P2
1
2
1
2
1
(a 96.5 A
Ê
; b 103.5 A
Ê
; c 113.6 A
Ê
) with two
F
ab
fragments in the asymmetric unit, which were independently re®ned
(®nal R-factor 21.7%). The resulting coordinates were used for a detailed
comparison with the modelled protein structure. It was found that the
mutual arrangement of the six complementarity-determining regions as
well as most of their backbone conformation had been correctly pre-
dicted. One major difference that was detected for the conformation of a
®ve residue insertion in complementarity-determining region L1 could be
explained by an erroneously de®ned segment in the structure of the anti-
body 4-4-20, which had been used as a template for this loop. In the light
of more recent crystallographic data it appears that this segment adopts
a new canonical structure. Apart from this region, most of the side-chains
in the antigen-binding site had been properly placed in the M41 model.
There was however one important exception concerning Trp H98, whose
side-chain conformation had been kept as it appeared in HyHEL-10. The
differing orientation of this residue in the model compared with the crys-
tal structure of the arti®cial F
ab
fragment M41 explains why an antigen
af®nity could not be detected so far. The detailed analysis of this and
other, more subtle deviations suggests how to make this F
ab
fragment
function by introducing a few additional amino acid changes into M41.
# 1997 Academic Press Limited
Keywords: antigen binding; computer modelling; immunoglobulin; protein
engineering; X-ray crystallography
*Corresponding author
Introduction
Attempts towards the rational design of proteins
with novel structures and functions are of interest
for two reasons. First, in basic science the com-
puter-aided construction of arti®cial proteins, to-
gether with their experimental characterization,
contributes to our understanding of the relation-
ship between folding, stability, and biochemical
activity in natural proteins and may lead to the
discovery of new principles in this respect. Second,
Abbreviations used: CDR, complementarity-
determining region; C
L
, constant domain of the light
chain; C
H
1, ®rst constant domain of the heavy chain;
DMSO, dimethyl sulphoxide; F
ab
, antigen-binding
fragment of antibody; F
v
, paired variable domains of
antibody heavy and light chains; NMR, nuclear
magnetic resonance; r.m.s., root-mean-square; RS, real
space; SA, simulated anealing; V
H
, variable domain of
the heavy chain; V
L
, variable domain of the light
chain.
J. Mol. Biol. (1997) 268, 934±951
0022±2836/97/200934±18 $25.00/0/mb971006 # 1997 Academic Press Limited