From the Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
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MN12H2 is a bactericidal antibody directed
against outer membrane protein PorA epitope P1.16 of Neisseria
meningitidis. Binding of MN12H2 to PorA at the meningococcal
surface activates the classical complement pathway resulting in
bacterial lysis. We have determined the crystal structure of the
unliganded MN12H2 Fab fragment in two different crystal forms and
compared it with the structure of the Fab in complex with a
P1.16-derived peptide. The unliganded Fabs have elbow bend angles of
155° and 159°, whereas the liganded Fab has a more closed elbow
bend of 143°. Substantial differences in quaternary and tertiary
structure of the antigen binding site are observed between the
unliganded and liganded MN12H2 Fab structures that can be attributed to
peptide binding. The variable light and heavy chain interface of the
liganded Fab is twisted by a 5° rotation along an axis approximately
perpendicular to the plane of the interface. Hypervariable loops H1,
H2, and framework loop FR-H3 follow this rotation. The hypervariable
loop H3 undergoes conformational changes but remains closely linked to
hypervariable loop L1. In contrast with the binding site expansion seen
in other Fab-peptide structures, the MN12H2 binding site is narrowed
upon peptide binding due to the formation of a "false floor"
mediated by arginine residue 101 of the light chain. These results
indicate that PorA epitope P1.16 of N. meningitidis is
recognized by the complement-activating antibody MN12H2 through induced
fit, allowing the formation of a highly complementary immune complex.
Antibody-antigen recognition is considered one of the most
specific intermolecular interactions in the immune system. In addition to their antigen binding specificity, antibodies display a variety of
secondary biological activities that are critical for host defense.
These include virus neutralization, complement activation, opsonization, and signal transduction. Structural aspects of these antibody-associated effector functions have been described for virus-neutralizing antibodies (1-4). The atomic details of the complexes between the Fab fragments of anti-viral antibodies and peptides derived from viral epitopes show common structural features: 1) binding of the peptides induces major rearrangements of the antibody
variable domains, and 2) the epitope-peptides bind in tight turn
conformations. For a bacterial epitope, we have recently found that it
was bound in a similar manner by complement-activating antibody MN12H2,
directed against outer membrane protein PorA of Neisseria
meningitidis (5).
PorA is a cation-selective transmembrane protein of 44 kDa that forms
trimeric pores in the meningococcal outer membrane. According to a
topology model, based on known porin structures, the protein is thought
to span the membrane in a 16-strand The first three-dimensional details of the bactericidal recognition of
PorA epitope P1.16 were unveiled in the crystal structure of the MN12H2
Fab fragment in complex with a peptide derived from PorA residues
180-187 (5). The fluorescein-labeled peptide was found in a type I
The capacity of meningococcal PorA to evoke antibodies that induce
complement-mediated bacterial killing has incited study to use this
protein as a target in vaccine development. In clinical vaccination
trials with outer membrane vesicles, it was shown that PorA was
critical for the induction of bactericidal antibodies in humans (9). It
was also shown that the presence of these antibodies correlate with
protection against meningococcal disease (10). The protective activity
of the immune complex between an antigen and a bactericidal antibody
depends on its ability to cross-link complement factor C1q. With the
binding of C1q, the classical complement pathway is activated, which
leads to the formation of a multiprotein membrane-attack complex,
causing bacterial membrane rupture.
Here, we present the three-dimensional structure of the unliganded
MN12H2 Fab fragment at 2.5 Å resolution based on two different crystal
forms and compare it with the Fab-P1.16 peptide complex. With both
structures available, we examine possible mechanisms by which
quaternary and tertiary changes following antigen binding may activate
the classical complement pathway.
Preparation of MN12H2 Fab Fragments--
The murine monoclonal
antibody MN12H2 was purified from hybridoma cell culture supernatant as
described previously (11). The Fab fragment was obtained by papain
digestion with papain-agarose beads (Sigma) at 37 °C for 4-16 h.
The digestion buffer consisted of 10 mM Tris-HCl (Fluka,
Buchs, Germany), pH 7.4, 1 mM EDTA, 0.02% w/v
NaN3, and 1 mM dithioerythritiol. After
digestion at least four prominent Fab isoforms could be identified by
isoelectric focusing (IEF)2
(Pharmacia Phast system, Pharmacia LKB, Uppsala, Sweden) with approximate pI values of 8.45, 8.65, 9.1 and 9.3. The IEF pattern of
MN12H2 Fab-peptide crystals showed that the crystallized Fab fragment
mainly consisted of the pI 8.65 isoform. Isolation of this isoform was
performed by anion exchange chromatography using a Q-Sepharose column
(Amersham Pharmacia Biotech). The Fab fraction of the papain digest was
loaded to the column using 20 mM
3-(cyclohexylamino)-1-propane sulfonic acid (CAPS) (Fluka), pH 9.8, 0.02% w/v NaN3 as a binding buffer. The different Fab
isoforms were eluted with a 0-0.15 M NaCl gradient. The
recovered isoforms were analyzed by SDS-polyacrylamide gel
electrophoresis, IEF, electron-spray mass spectrometry, and dynamic
light scattering using a DynaPro-801 dynamic light scattering instrument (Protein Solutions Ltd., High Wycombe, Buckinghamshire, United Kingdom). Specific binding of the purified pI 8.65 Fab isoform
to the P1.16 epitope was determined by means of fluorescence polarization experiments with a synthetic epitope peptide, as described
earlier (8).
Crystallization and Data Collection--
Preliminary
crystallization conditions of the unliganded MN12H2 Fab were identified
with a set of screening solutions using concentrations of 15-45% v/v
2-methyl 2,4 peptanediol (MPD) (Fluka) and 5-25% w/v polyethylene
glycol 3000 (Fluka) as precipitating agents in combination with low
molar concentrations of CaCl2, MgCl2, and
CdCl2. The screening solutions were buffered using sodium
acetate, 2-morpholinoethane-sulfonic acid (MES) (Fluka), HEPES (Fluka),
or Tris-HCl (Fluka) with pH values of 4.5, 6.5, 7.5, and 8.5, respectively. Crystals for data collection were obtained at 4 °C in
hanging drops using 20-30% v/v MPD and 20 mM
CdCl2 in 50 mM MES buffer, pH 6.7.
Data collection was performed at 120 K on a McScience DIP-2020 image
plate detector using graphite monochromatized CuK
Data were collected from two different Fab crystal forms obtained under
slightly different conditions (27 and 20% (v/v) MPD). Crystals grown
at 27% MPD were C-centered orthorhombic C2221 with cell
dimensions a = 86.0, b = 114.9, c = 153.1 Å. Diffraction to about 3.2 Å resolution
was collected to 100% completeness from a single crystal of
approximate size 0.2 × 0.2 × 0.15 mm3. The
asymmetric unit contains one Fab molecule with a Matthews coefficient
(Vm) (13) of 3.8 Å3/Da and a
solvent content of 68%. The 20% MPD crystals belonged to the
C-centered monoclinic space group C2, with unit cell dimensions a = 114.6, b = 85.9, c = 87.1 Å and Molecular Replacement and Refinement--
The structures of the
unliganded MN12H2 Fabs were solved by molecular replacement using the
MN12H2 Fab of Protein Data Bank entry 1MPA as a search model. The
rotation function, the Patterson correlation function optimizing the
relative orientation of the four Fab domains (VL,
VH, CL, and CH1), the translation
function, and structure refinement were performed with the program
Crystallography and NMR System (14-17). Refinement used the maximum
likelihood target with all experimental amplitudes except for a
randomly selected test set of 10% that was used for cross-validated
The structure of the MN12H2 Fab in complex with the P1.16-derived
fluorescein-conjugated peptide was also subjected to the maximum
likelihood target and torsion angle dynamics refinement method.
Comparison of the Structures--
As an indication of changes in
the quaternary structure upon ligand binding for each molecule the
angle between the pseudo-2-fold rotation axes of the
VL-VH and CL-CH1
domains and the V and C superdomains (elbow bend angle) was determined
using the program ROTMOL (kindly supplied by J. N. Herron). To further
analyze quaternary and tertiary changes, the structures of the
unliganded Fab and Fab-peptide complex were overlaid by least squares
rotation and translation using O (18).
Structure Determination of the Unliganded Fab--
The structure
of the unliganded Fab was solved for two different crystal forms. The
monoclinic crystal form (space group C2) diffracted to 2.5 Å resolution, whereas the orthorhombic crystal form (space group
C2221) diffracted to a resolution of 3.2 Å. Crystallization and data collection statistics are given in Table I.
Structures were refined to an R factor of 23% and an Rfree
of 27% for the monoclinic crystal form and to an R factor of 25% and
an Rfree of 30% for the orthorhombic crystal form.
Geometric parameters evaluated with PROCHECK (19) and WHAT IF (20) show acceptable values for both crystal structures. Refinement and model
statistics are given in Table II. Almost
90% of the residues of the monoclinic crystal form of the unliganded
Fab had main chain torsion angles that fell within the energetically
most favored regions of the Ramachandran plot (21), whereas none were
found in the disallowed regions (Fig.
1A).
The final unliganded MN12H2 Fab model in the monoclinic crystal form
contains all 219 light chain amino acid residues and 221 of the 225 residues of the heavy chain as determined by electron spray mass
spectrometry (molecular mass, 49,201 Da). The model also includes the
classically disordered interchain-disulfide region Cys-136H-Gly-141H
(Fig. 1B), for which good density was observed. Some basic
residues at the exterior of the Fab display ill-defined electron
density (79L, 82L, 86L, 108L, 56H, 63H, and 69H) and the side chain
conformation of Phe-106H at the top of hypervariable loop H3 must also
be considered as tentative. In the orthorhombic unliganded Fab the
interchain-disulfide region residues Gly-137H-Gly-141H were excluded
from the model due to weak electron density. A 4° difference in elbow
bend angle was found between the two unliganded Fab crystal forms
(Table III). Otherwise, both structures
are virtually identical.
As in the MN12H2 Fab-peptide complex (5), strong electron density peaks
were seen in both unliganded Fab crystal forms. Because cadmium ions
were essential for crystallization and because of the vicinity of
putative cadmium binding residues, the positions of these 5 Maximum Likelihood Refinement of the 1MPA Structure--
Because
the structures of the free and complexed MN12H2 Fab were refined using
different techniques, for good comparison of both models, the
Fab-peptide structure (Protein Data Bank entry 1MPA) was subjected to
refinement using the maximum likelihood target. Refinement and
rebuilding of heavy chain regions 42H-43H, 136H-141H and 156H-158H
resulted in a drop in free R factor by 4.5% from 30.9 to 26.4%.
Rebuilding of these heavy chain regions also improved their abnormal
main chain Comparison of the Free and Complexed MN12H2 Fab Structures--
We
observed elbow bend angles of 155° and 159° for the unliganded Fab
in the monoclinic crystal form and the orthorhombic crystal form,
respectively. For the liganded MN12H2 Fab, a more closed elbow bend was
found with an angle of 143° between the pseudo-2-fold rotation axis
of the V and C superdomains (Table III).
A 5° rotation was observed between the variable domains of the
unliganded Fabs and the Fab-peptide complex, along an axis approximately perpendicular to the VL-VH
interface. As illustrated in Figs. 2 and
3A, the largest coordinate
differences resulting from this rotation were found at the tips of the
hypervariable loops (C
Major side chain displacements between the unliganded Fabs and the
Fab-peptide complex were observed in loops L3 and H3. In both
unliganded Fabs, arginine residue 101L protrudes from the floor of the
antigen binding site (Fig. 3C), thereby creating a
positively charged bulge. This is also illustrated in the molecular surface of the unliganded antigen binding site in Fig.
4A, colored for electrostatic
potential. In contrast, Arg-101L spans the binding pocket in the
Fab-peptide complex, and its guanidinium group forms hydrogen bridges
with residues Ser-97H and Tyr-41L. In the newly refined liganded Fab
structure, additional interactions are seen with Arg-101L: a
water-mediated hydrogen-bonding interaction with the hydroxyl group of
Ser-94L (hypervariable loop L3) and a hydrogen bond with N Crystal Contacts--
Between both free Fab crystal forms and the
Fab-peptide complex, the hypervariable loops play different roles in
crystal packing interactions. In the monoclinic as well as the
orthorhombic unliganded Fab L3 loop, residue His-98L is involved in
head-to-tail symmetry interactions with the C terminus of the light
chain, mediated by a cadmium ion (defining the variable domain as head
and the constant domain as tail). In the monoclinic crystal form, 70% of all hypervariable loop crystal contacts involve additional head-to-tail van der Waals interactions between the tips of loops L1
and L2 and the interchain disulfide region. Also, several salt links
with the C-terminal region of the CH1 domain of a
symmetry-related molecule are observed. The interactions between loop
L1 and the interchain disulfide residues Gly-137H and Thr-140H in the
monoclinic unliganded Fab result in unexpectedly well defined electron
density for this archetypically disordered region. In the orthorhombic Fab, no interactions are observed between the three light chain hypervariable loops and the interchain disulfide region. In both unliganded Fab structures, the H3 loop is packed head-to-head with the
binding site of a symmetry-related molecule.
In the complexed Fab, crystal contacts mainly involve head-to-head
packing between the apices of hypervariable loop L1 and loops H1 and H3
of a symmetry-related molecule. The fluorescein label of the peptide
accounts for 11 additional head-to-tail contacts with the C terminus of
the CH1 domain. A single interaction is observed between
loop L1 and the interchain disulfide region. In contrast with the free
Fab crystal forms, the liganded Fab displays crystal contacts with
elbow bend residues 112L, 113L, and 114L that are packed against the
heavy chain of a symmetry-related molecule.
Comparison of two crystal forms of the unliganded MN12H2 Fab with
the liganded structure of the epitope-peptide Fab complex reveals
significant conformational changes. The surface representations of the
binding sites of both structures in Fig. 4 indicate the quaternary and
tertiary changes of the Fab upon peptide binding. The antigen binding
site of MN12H2 may be thought of as a left-handed baseball glove, with
hypervariable loops L1 and L3 forming the thumb, and the VH
domain (with loops H3, H2, and H1) as the fingers. As in the baseball
glove, the thumb (loop L1) and the forefinger (loop H3) remain
connected upon peptide binding through a "two-bar" web formed by a
tandem of tyrosine-aspartate interactions, whereas the other fingers
(loops H2, framework region-H3, and H1) follow the shape of the peptide
(see also Fig. 2). In the unliganded Fab, arginine residue 101L forms a
positively charged bulge in the binding cavity. Upon peptide binding,
this bulge is depressed, and its charge is neutralized due to the
formation of a false floor that narrows the cavity and connects four
hypervariable loops (L1, L3, H1, and H3). The side chain rearrangements
in the H3 loop represented by tyrosines 100H and 103H are necessary to clear the binding site to further accommodate the peptide binding. When
the P1.16 peptide is positioned in the binding site of the unliganded
Fab, the side chain of these residues sterically blocks the binding of
the peptide.
The sterical hindrance of hypervariable loop H1 residues that is
encountered by the peptide is overcome by the observed 5° twist of
the VL-VH interface in the liganded Fab. This
rotation brings H1 residue Tyr-33H in position to form a hydrophobic
stack with peptide residue Thr-183P. The VL-VH
twist also decreases the distance between the C With the exposed guanidinium moiety of Arg-101L in the cavity
surrounded by basic residues His-31L and Arg-59L, an overall positively
charged binding site is formed in the unliganded Fab, as illustrated in
Fig. 4A. Although Arg-101L does not contribute directly to
peptide binding, it could explain the medium-affinity cross-reactive
binding mode of MN12H2 for negatively charged self-antigens such as
single stranded DNA and
cardiolipin.3 The appearance
of basic residues at these sites within the light chain hypervariable
loops and especially at the junction between V Relative disposition of VL-VH domains has been
observed before in Fab structures upon binding of a peptide (1, 24,
25). It illustrates the intrinsic flexibility of antibodies in adapting to the shape of an antigen. The largest VL-VH
rotations have been observed for anti-DNA Fab BV0-401 (7.5°) (26)
and for anti-HIV Fab 50.1 upon complexation with a V3 loop peptide
(16°) (25). Unlike MN12H2, these Fab structures show a large decrease
in the number of VL-VH interface contacts upon
complexation with their antigen. The loss of self-contacts is mainly
ascribed to rearrangements of the H3 loop, which moves out of the
binding site with root mean square deviations in backbone atoms up to 5 Å. In contrast with this binding site expansion, the MN12H2 Fab shows
a closer association of both domains upon peptide binding by forming a false floor. The 5° rotation of the H1 and H2 loop narrows the cavity
even more and holds the peptide in a tight lock. Because the backbone
of the H3 loop remains fixed, massive side-chain rearrangements of
tyrosines 100H and 103H are essential for the binding site to adapt to
the shape of the peptide.
All of the structural changes mentioned in this section appear to be
necessary for peptide binding. Because both monoclinic and orthorhombic
crystal forms of the unliganded Fab display similar features in a
different crystal packing environment, the conformational differences
with the liganded Fab are believed to be peptide-induced. No
conformational changes of fluorescein binding residue Arg-59H have been
detected between the unliganded Fabs and 1MPA, suggesting no or only
minor effects of the fluorescein molecule on the shaping of the binding site.
Conformational changes in the elbow bend angle and the distal
extremities of the constant region, such as the interchain disulfide region, are believed to be induced by the general flexibility of these
regions and by their different crystal packing interactions. The elbow
bend regions at the junction between the variable and constant domains
are very flexible and there have been differences found in the elbow
bend angle between unliganded structures as well as liganded structures
of the same Fab (27). Stanfield et al. (25) even reported a
13° difference in elbow bend angle between two complexed 50.1 Fab
molecules found in the same asymmetric unit, also illustrating this
general flexibility.
There is no evidence that the peptide-induced conformational changes,
other than adjusting fit and complementarity to the antigen, provide a
signal that is transmitted to the constant domains triggering secondary
biological activities. The accessibility and the tight-turn recognition
motif of immunogenic loops might be important for evoking antibodies
that are able to neutralize viral infections or activate the complement
system. Several mechanisms have been postulated for virus neutralizing
activities of antibodies, including interference with the viral
attachment to the cell and viral internalization, induction of
structural changes as a consequence of binding, and intracapsid
cross-linking, preventing uncoating of the virus (28). A mechanism for
the complement activating capacity of bactericidal antibody MN12H2 may
be the improved complementarity between the P1.16 epitope and the
binding site. This tight conformational state might decrease the
off-rate of the complex, thereby augmenting the effective antibody
valence on the bacterial surface for cross-linking C1q.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-barrel conformation (6). The
model predicts eight extended extracellular loops. Two of these surface
exposed loops (loops 1 and 4) are highly immunogenic and evoke
antibodies that induce complement mediated bacterial killing. MN12H2 is
a bactericidal antibody that is elicited against loop 4 of PorA. The
recognition of loop 4 epitope P1.16 by MN12H2 is used as a model for
studying the molecular and structural details of bactericidal antibody
recognition of PorA.
-turn conformation in the antigen binding cavity. The structure
revealed several hydrophobic and electrostatic interactions between
both binding partners, including a salt bridge between aspartate 182P
of PorA and MN12H2 light chain residue histidine
31L.1 With the results from a
thermodynamic study, this salt bridge was identified as the key
interaction explaining the increased incidence of meningitis in United
Kingdom in the early 1980s, caused by a D182N mutant strain of N. meningitidis (7, 8).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
radiation from a Nonius FR570 rotating anode (Nonius, Delft, The Netherlands) operated at 45 mV and 95 mA. Diffraction data were auto-indexed and processed with DENZO and SCALEPACK (12).
= 122.7°. A complete data set was collected from a
single crystal with dimensions of 0.2 × 0.2 × 0.15 mm3 diffracting to 2.5 Å resolution. The monoclinic
crystal form also contains one Fab in the asymmetric unit,
Vm is 3.6 Å3/Da, with a solvent
content of 66%. Crystallization and data collection details are
summarized in Table I.
A weighting. The automated refinement procedure consisted of
conjugate minimization followed by torsion angle molecular dynamics in
combination with simulated annealing. Refinement was preceded by
calculation of a bulk-solvent model, estimate
A, and weight values.
Rounds of refinement were followed by rebuilding of the models in O
(18) using
A weighted maps. Finally, the unliganded Fab models were subjected to restrained B-factor refinement.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Crystallization and data collection
Refinement/statistics
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Fig. 1.
A, Ramachandran plot of the unliganded
MN12H2 Fab. Shown is the main chain and
torsion angle plot of
the unliganded Fab structure in the monoclinic crystal form as produced
by PROCHECK (19). 89% of the free Fab non-glycine residues are found
in regions with most favored geometry for
-helices,
-sheets, and
loops (A, B, and L), shaded with the
darkest gray. Residues Val-56L and Ser-180H are found in
generously allowed regions (~b, ~l, and
~p). B, stereo pair of a
a-weighted
2Fo
Fc electron
density map of the CH1 interchain-disulfide region.
Pseudodyad and elbow bend angles for the unliganded and
peptide-complexed MN12H2 Fab
electron density peaks are very likely to be occupied by cadmium ions.
As in the 1MPA structure, a putative cadmium binding site was found
near the N
1 of His-98L. In the monoclinic unliganded Fab structure,
this cadmium is coordinated by two water molecules, as in the 1MPA
structure, and by the carboxylate oxygens of Glu-218L at the N terminus
of a symmetry related molecule. A second cadmium ion interacts with the
N
2 of His-172H, the N
2 of Asn-143L, and a water molecule. The
third cadmium is coordinated by the carboxylate oxygens of Glu-190L,
the N
2 nitrogen of His-194L, and a water molecule and via crystal
packing interactions with the carboxylate moiety of a symmetry-related
Asp-181H. In the orthorhombic crystal form, an additional cadmium ion
was observed that occupied a special position situated on a 2-fold
rotation axis parallel to b. The ion was found to be
coordinated by the carboxylate oxygens of four glutamate residues,
including Glu-84L, Glu-86L, and their symmetry-related residues.
However, some of the densities assigned to coordinating water molecules
may actually be occupied by chloride ions.
and
torsion angles as indicated by the Ramachandran
plot (21). Structure validation of the model confirm these results
showing improved model geometry, as indicated by a decrease in bond and
angle violations and a reduction of close contacts (see also Table
IV). Because of ambiguous density,
C-terminal residue Ile-225H was removed from the model. No significant
conformational differences were observed for the bound
fluorescein-labeled peptide and the antigen binding site.
Refinement statistics of liganded Fab
-coordinate differences up to 3.6 Å for hypervariable loop H1 and up to 3.3 Å for H2). The H3 loop does
not follow this domain rotation, and its backbone is relatively kept in
position with only minor C
-coordinate differences from
0.2 Å for Asp-105H and up to 1.6 Å for Ala-104H. As depicted in Fig.
3B, the H3 loop is fixed to loop L1 by a tandem of Tyr-Asp
hydrogen bonds. The side chain hydroxyls of tyrosine residues 37L and
41L bind to the carboxylate oxygens of aspartate residues 102H and 109H
of loop H3. These VH-VL interface interactions
are further tightened by a hydrogen bond between the N
of
hypervariable loop L2 residue Lys-55L and the carbonyl oxygen of
Phe-101H (loop H3).
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Fig. 2.
C displacements of the
VH chain between the unliganded MN12H2 Fab and the
Fab-peptide complex. For this analysis, the corresponding
VL chains of both Fab structures were overlaid using the
following sets of C
atoms corresponding to segments of
the conserved
-pleated sheet regions (framework regions) as defined
by Kabat et al. (29): VL, 19L-25L, 37L-43L,
67L-72L, 75L-80L, 90L-95L, 102L-108L; VH, 18H-24H,
33H-39H, 68H-73H, 78H-83H, 93H-98H, 110H-116H.
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Fig. 3.
A, relative positions of the variable
light and heavy chains in the dimers of the unliganded Fab and the
Fab-peptide complex. B, conformational differences in hyper
variable loop H3 between the unliganded MN12H2 Fab and the Fab-peptide
complex. C, superposition of the cavity floor residues of
the unliganded and liganded MN12H2 Fabs. For these figures the
corresponding VL chains of the monoclinic unliganded Fab
and the liganded Fab structures were overlaid as a dimer as described
in Fig. 2. Light and heavy chain residues of the liganded Fab are shown
in green and magenta. Unliganded Fab residues and
their corresponding backbone positions are shown in yellow.
These figures were produced with MOLSCRIPT and RASTER3D (30, 31).
2 of
His-35H (hypervariable loop H2). The largest atomic displacements
between the free and the complexed Fab were observed in the H3 loop. As
illustrated in Figs. 3B and 4A, a dramatic
displacement was seen for Tyr-103H with a 9-Å difference of the
hydroxyl oxygens between both structures. The hydroxyl oxygen of
Tyr-100H, pointing toward the binding site in the unliganded Fab, is
moved away 3.4 Å from the cavity in the Fab-peptide complex and points
upward.
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Fig. 4.
Molecular surface areas of the binding site
of the free MN12H2 Fab (A) and the Fab-peptide complex
(B). The surface is colored for electropotential
(blue for positive charge, red for negative
charge). The approximate locations of hypervariable loops and binding
site residues are indicated. This figure was prepared with the program
GRASP (32).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
atoms of
hypervariable loops L1 (Arg-101L) and H3 (Ser-97H) from 13 to 11.5 Å,
thereby facilitating the formation of a Arg-101L-mediated bidentate
hydrogen bond with the side chain hydroxyl oxygen of Ser-97H and the
hydroxyl oxygen of Tyr-41L. The hydrogen bond between the N
2 group
of Arg-101L and N
2 of His-35H (hypervariable loop H2) induces an
almost 90° rotation of the 35H imidazolium ring bringing the N
2
nitrogen in position to fix the peptide backbone at the newly formed
cavity floor. The formation of this false floor, directed by Arg-101L, stabilizes the topography of the complexed
VL-VH dimer by locking four hypervariable loops
(L1, L3, H1, and H3) into a new conformation. A thermodynamic study on
the interaction with MN12H2 and the P1.16 peptide revealed that these
interactions, together with the release of structured water from the
binding pocket and the newly formed interactions between Fab and
peptide, favor the interaction by 100 kJ mol
1 (8). It
largely offsets the 50 kJ mol
1 cost in free energy needed
to induce the structural changes in both binding partners (as
determined by the analysis of Sturtevant (22)).
and
J
1 genes (101L) fits the general pattern of DNA-binding antibodies described by Radic and Weigert (23). Whether the binding of
these autoantigens adopt different complexed structures remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank The Dutch National Institute of Public Health and the Environment in Bilthoven for providing the MN12H2 hybridoma cell-culture supernatant. We also thank Dr. J. N. Herron from the University of Utah for critically reviewing the manuscript. The technical assistance of C. Versluis from the Utrecht University with the electron-spray mass spectrometry experiments is gratefully acknowledged.
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FOOTNOTES |
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* This research was financially supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U60442 (MN12H2 variable light chain) and U60443 (MN12H2 variable heavy chain).
The atomic coordinates and structure factors (codes 1MPA and 2MPA) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed: Tel.: 31-30-2533502;
Fax: 31-30-2533940; E-mail: p.gros{at}chem.uu.nl.
The abbreviations used are:
IEF, isoelectric
focussing; CAPS, 3-(cyclohexylamino)-1-propane sulfonic acid; CH1, constant heavy domain 1; CL, constant
light domain; MES, 2-morpholinoethanesulfonic acid; MPD, 2-methyl
2,4-peptanediol; VH, variable heavy domain; VL, variable light domain; Vm, Matthews coefficient; V, variable region
; J
, joining region
.
1 Amino acid residues of the MN12H2 heavy chain and light chain are indicated by H and L, respectively. The peptide residues are indicated by P. Throughout the text, a strict sequential numbering system is used.
3 J. M. H. van den Elsen, unpublished results.
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