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PDBsum entry 2nz9
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Toxin/immune system
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PDB id
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2nz9
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Contents |
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1267 a.a.
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216 a.a.
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217 a.a.
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References listed in PDB file
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Key reference
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Title
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Molecular evolution of antibody cross-Reactivity for two subtypes of type a botulinum neurotoxin.
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Authors
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C.Garcia-Rodriguez,
R.Levy,
J.W.Arndt,
C.M.Forsyth,
A.Razai,
J.Lou,
I.Geren,
R.C.Stevens,
J.D.Marks.
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Ref.
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Nat Biotechnol, 2007,
25,
107-116.
[DOI no: ]
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PubMed id
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Abstract
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Broadening antibody specificity without compromising affinity should facilitate
detection and neutralization of toxin and viral subtypes. We used yeast display
and a co-selection strategy to increase cross-reactivity of a single chain (sc)
Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that
binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype
with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to
87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the
molecular basis for improved cross-reactivity, we determined the X-ray
co-crystal structures of wild-type and cross-reactive antibodies complexed to
BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution
of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding.
The results show how an antibody can be engineered to bind two different
antigens despite structural differences in the antigen-antibody interface and
may provide a general strategy for tuning antibody specificity and
cross-reactivity.
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Figure 2.
Figure 2. Overview and specific interactions of the CR1-BoNT/A1
co-crystal. (a) Overall view of BoNT/A1 (yellow) in complex
with the CR1 Fab with its light and heavy chains in magenta and
green, respectively. (b) Overview of the CR1-BoNT/A1 interface,
with the antigen contacting loops (H1, H2, H3, L1, L2 and L3)
and toxin  -strands
indicated. (c) Detailed view of contacts between CR1 Fab and
BoNT/A1. A cartoon representation of BoNT/A1 is shown with
carbons (yellow), nitrogens (blue) and oxygens (red). Amino acid
contacts are indicated by magenta (V[L]), green (V[H]) and black
(BoNT/A) numbering. V[L], variable light chain; V[H], variable
heavy chain.
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Figure 3.
Figure 3. Location of interactions that differ between CR1 and
BoNT/A1 and BoNT/A2 and affect differential BoNT/A binding.
(a) Location of CR1-BoNT/A1 contact residues and residues
differing between BoNT/A1 and BoNT/A2. The alignment of BoNT/A1
and A2 subtypes shows strict sequence conservation in white
letters on red background, and strong sequence conservation in
red letters. The residues composing the CR1 epitope of the H[CN]
lectin (residues 874–1094) and H[CC] trefoil (residues
1095–1295) subdomains are indicated with red triangles,
energetically important residues are shown with black triangles.
Disulfide bonds are indicated using green numbers. The secondary
structure elements of the BoNT/A1 binding domain structure are
labeled  (
-helix),
(
-strand)
and TT (turn). (b) Structural location of differences between
BoNT/A1 and A2 and impact on CR1 interactions. (i,ii) Surface
representations of BoNT/A1 (yellow) in complex with CR1 (V[L] in
magenta and V[H] in green) showing patches of sequence
variability between BoNT/A1 and BoNT/A2 subtypes in slate blue.
(iii,iv) Close-up view of sequence variability between T1063 and
H0164 of BoNT/A1 (yellow, iii) and modeled P1063 and R0164 of
BoNT/A2 (cyan, iv) in complex with CR1 (V[L] in magenta and V[H]
in green). (v,vi) Surface representations of BoNT/A1 (yellow)
with BoNT/A1 and BoNT/A2 sequence differences in slate blue. Key
differences between BoNT/A1 and BoNT/A2 that are functionally
important for binding (high  G
values) are shown in dark blue (1063 and 1064). Functionally
important BoNT/A residues (high G
values) that do not differ between BoNT/A1 and BoNT/A2 are shown
in red. Panel v shows CR1 with its light and heavy chains in
magenta and green, respectively, with its H1 loop in tan. Panel
vi is looking down onto the CR1 epitope of BoNT/A1 with CR1
removed. (c) Details of the interaction between AR2 and CR1 and
BoNT/A1 and A2 at the H1 loop. Close-up view of the H1 loop
showing sequence and structural differences between BoNT/A1
(yellow) and BoNT/A2 (cyan), in complex with the CR1 (green) and
AR2 (orange) Fabs. The BoNT/A2-CR1 and BoNT/A2-AR2 structures
are modeled.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Biotechnol
(2007,
25,
107-116)
copyright 2007.
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