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PDBsum entry 1wej
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Complex (antibody/electron transport)
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PDB id
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1wej
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Contents |
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214 a.a.
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223 a.a.
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105 a.a.
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural basis for the binding of an anti-Cytochrome c antibody to its antigen: crystal structures of fabe8-Cytochrome c complex to 1.8 a resolution and fabe8 to 2.26 a resolution.
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Authors
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S.E.Mylvaganam,
Y.Paterson,
E.D.Getzoff.
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Ref.
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J Mol Biol, 1998,
281,
301-322.
[DOI no: ]
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PubMed id
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Abstract
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A complete understanding of antibody-antigen association and specificity
requires the stereochemical description of both antigen and antibody before and
upon complex formation. The structural mechanism involved in the binding of the
IgG1 monoclonal antibody E8 to its highly charged protein antigen horse
cytochrome c (cyt c) is revealed by crystallographic structures of the
antigen-binding fragment (Fab) of E8 bound to cyt c (FabE8-cytc), determined to
1.8 A resolution, and of uncomplexed Fab E8 (FabE8), determined to 2.26 A
resolution. E8 antibody binds to three major discontiguous segments (33 to 39;
56 to 66; 96 to 104), and two minor sites on cyt c opposite to the exposed haem
edge. Crystallographic definition of the E8 epitope complements and extends
biochemical mapping and two-dimensional nuclear magnetic resonance with
hydrogen-deuterium exchange studies. These combined results demonstrate that
antibody-induced stabilization of secondary structural elements within the
antigen can propagate locally to adjacent residues outside the epitope.
Pre-existing shape complementarity at the FabE8-cytc interface is enhanced by 48
bound water molecules, and by local movements of up to 4.2 A for E8 antibody and
8.9 A for cyt c. Glu62, Asn103 and the C-terminal Glu104 of cyt c adjust to fit
the pre-formed VL "hill" and VH "valley" shape of the
grooved E8 paratope. All six E8 complementarity determining regions (CDRs)
contact the antigen, with CDR L1 forming 46% of the total atomic contacts, and
CDRs L1 (29%) and H3 (20%) contributing the highest percentage of the total
surface area of E8 buried by cyt c (550 A2). The E8 antibody covers 534 A2 of
the cyt c surface. The formation of five ion pairs between E8 and flexible cyt c
residues Lys60, Glu62 and Glu104 suggests the importance of mobile regions and
electrostatic interactions in providing the exquisite specificity needed for
recognition of this extremely conserved protein antigen. The highly homologous
VL domains of E8 and anti-lysozyme antibody D1. 3 achieve their distinct
antigen-binding specificities by expanding the impact of their limited sequence
differences through the recruitment of different sets of conserved residues and
distinctly different CDR L3 conformations.
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Figure 1.
Figure 1. Stereo views of FabE8-cyt c complex and FabE8
X-ray structures. a, Ribbon representation of the FabE8-cyt c
interface region. E8 V-domain (V[L], white; V[H], yellow) and
cyt c (orange). Highlighted are the three major discontiguous
segments of E8 epitope on cyt c (33 to 39, 56 to 66 and 96 to
104 in red) and the six E8 CDRs (L1 and H1, red; L2 and H2,
yellow; L3 and H3, green). b, Ribbon representation of the FabE8
(pink) and FabE8-cyt c (L, white; H, yellow; cyt c, orange)
structures with V-domains superimposed. The elbow angles of the
two structures differ by 6°. c, Cyt c (orange) bound to
CDR-H3 of FabE8-cyt c (yellow), with CDR-H3 of D1.3Fv-HEL
(purple) superimposed. The tip of E8 H3 (top) complements the
second 30 s type II b-turn (35 to 38) of cyt c. E8 Phe91L and
Trp96L of L3 (white) and Tyr33H1 (yellow) neighbour E8 H3. The
base of E8 H3 is in the extended conformation and the
carboxylate group of Asp105H3[101] hydrogen bonds with the
Trp107H[103] FR4 and Tyr36L FR2 side-chains. In the D1.3
antibody (purple), the base of H3 is "kinked" at position [101].
The Asp side-chain at this position is flipped 180° with
respect to E8 Asp105H3[101] and ion pairs with ArgH3[94]
(equivalent position Gly98H in E8). Oxygen and nitrogen atoms
are shown as red and blue spheres, respectively and hydrogen
bonds as yellow dotted lines.
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Figure 3.
Figure 3. Stereo views of buried surface areas and residues
of the FabE8-cyt c (a) paratope with 48 interface water
molecules, and (b) E8 epitope. Views are 90° fromFigure 1a.
Water molecules (green spheres) form a collar around the buried
surface areas of E8 antibody (pink mesh) and cyt c (blue mesh).
C^a traces of E8 V[L] are shown in white, V[H] in yellow and cyt
c in orange. Side-chains in the E8 paratope are displayed and
labelled with CDRs L1 and H1 in red, L2 and H2 in yellow and L3
and H3 in green. The E8 epitope consists of three major
discontiguous segments (33 to 39; 56 to 66 and 96 to 104) shown
with magenta side-chains and two minor sites composed of the
N-terminal acetyl group (Ac), Val3 (top) and Lys22 (left) (with
white side-chains and yellow labels) at the periphery. Arg38 and
Tyr74 (white side-chains and green labels) lie outside the E8
epitope. Oxygen and nitrogen atoms are shown as red and blue
spheres, respectively.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1998,
281,
301-322)
copyright 1998.
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Secondary reference #1
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Title
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Biochemical implications from the variable gene sequences of an anti-Cytochrome c antibody and crystallographic characterization of its antigen-Binding fragment in free and antigen-Complexed forms.
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Authors
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S.E.Mylvaganam,
Y.Paterson,
K.Kaiser,
K.Bowdish,
E.D.Getzoff.
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Ref.
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J Mol Biol, 1991,
221,
455-462.
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PubMed id
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Secondary reference #2
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Title
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Monoclonal antibodies to horse cytochrome c expressing four distinct idiotypes distribute among two sites on the native protein.
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Authors
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F.R.Carbone,
Y.Paterson.
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Ref.
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J Immunol, 1985,
135,
2609-2616.
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PubMed id
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