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PDBsum entry 3i9g
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Immune system
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PDB id
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3i9g
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References listed in PDB file
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Key reference
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Title
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The crystal structure of sphingosine-1-Phosphate in complex with a FAB fragment reveals metal bridging of an antibody and its antigen.
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Authors
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J.M.Wojciak,
N.Zhu,
K.T.Schuerenberg,
K.Moreno,
W.S.Shestowsky,
M.Hiraiwa,
R.Sabbadini,
T.Huxford.
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Ref.
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Proc Natl Acad Sci U S A, 2009,
106,
17717-17722.
[DOI no: ]
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PubMed id
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Abstract
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The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant
roles in angiogenesis, heart disease, and cancer. LT1009 (also known as
sonepcizumab) is a humanized monoclonal antibody that binds S1P with high
affinity and specificity. Because the antibody is currently in clinical trials,
it is important to confirm by structural and biochemical analyses that it binds
its target in a predictable manner. Therefore, we determined the structure of a
complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 A
resolution. The antibody employs unique and diverse strategies to recognize its
antigen. Two metal ions bridge complementarity determining regions from the
antibody light chain and S1P. The coordination geometry, inductively coupled
plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical
assays suggest that these are Ca(2+). The amino alcohol head group of the
sphingosine backbone is recognized through hydrogen bonding interactions from 1
aa side chain and polypeptide backbone atoms of the antibody light and heavy
chains. The S1P hydrophobic tail is almost completely enclosed within a
hydrophobic channel formed primarily by the heavy chain. Both treatment of the
complex with metal chelators and mutation of amino acids in the light chain that
coordinate the metal atoms or directly contact the polar head group abrogate
binding, while mutations within the hydrophobic cavity also decrease S1P binding
affinity. The structure suggests mechanistic details for recognition of a
signaling lipid by a therapeutic antibody candidate. Moreover, this study
provides direct structural evidence that antibodies are capable of using metals
to bridge antigen:antibody complexes.
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Figure 2.
X-ray crystal structure of the LT1009 Fab:S1P complex. (A) A
ribbon diagram representation of the antibody Fab fragment. The
light chain is colored gold and the heavy chain is brown. S1P is
depicted as a ball-and-stick model with cpk coloring and the 2
Ca^2+ ions are represented as light gray spheres. (B) Close-up
view of the S1P binding site. Electron density from a 2 F[O] -
F[C] refined map contoured at 1.1 Ļ is displayed for the region
of the model within 1.9 Å of each atom in S1P. (C) A
semitransparent surface representation of the Fab reveals the
extent to which S1P, represented as cpk spheres, is buried upon
binding to LT1009.
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Figure 5.
(A) Overlay of LT1009 and Q425 anti-CD4 antibody Fab crystal
structures illustrates their overall homology and different
Ca^2+ binding sites. LT1009 is colored as in previous figures
while the Q425 light chain is cyan and the heavy chain fragment
is blue. For the sake of clarity, S1P has been removed from the
LT1009 Fab model. (B) A close up view of Ca^2+ binding sites in
the 2 models reveals conservation of metal coordinating side
chains and the different conformations of CDR-H3. (C) Schematic
representation of a possible mechanism of S1P binding by LT1009.
In its unbound conformation, the light chain (labeled āLā in
the figure) binds one Ca^2+ at a site similar to that observed
in the Q425 antibody structures. S1P binding introduces
conformational changes that disrupt the original Ca^2+ binding
motif as well as potentially introducing one additional Ca^2+.
The phosphate group of S1P then combines with 2 Ca^2+ to produce
the extremely stable Ca^2+ coordination complex observed in the
S1P:LT1009 Fab complex crystal structure.
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Secondary reference #1
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Title
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Production and characterization of monoclonal anti-Sphingosine-1-Phosphate antibodies.
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Authors
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N.O'Brien,
S.T.Jones,
D.G.Williams,
H.B.Cunningham,
K.Moreno,
B.Visentin,
A.Gentile,
J.Vekich,
W.Shestowsky,
M.Hiraiwa,
R.Matteo,
A.Cavalli,
D.Grotjahn,
M.Grant,
G.Hansen,
M.A.Campbell,
R.Sabbadini.
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Ref.
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J Lipid Res, 2009,
50,
2245-2257.
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PubMed id
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