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PDBsum entry 2vh5
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Immune system
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PDB id
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2vh5
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Contents |
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114 a.a.
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104 a.a.
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166 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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Functional intracellular antibody fragments do not require invariant intra-Domain disulfide bonds.
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Authors
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T.Tanaka,
T.H.Rabbitts.
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Ref.
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J Mol Biol, 2008,
376,
749-757.
[DOI no: ]
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PubMed id
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Abstract
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Intracellular antibody fragments that interfere with molecular interactions
inside cells are valuable in investigation of interactomes and in therapeutics,
but their application demands that they function in the reducing cellular
milieu. We show here a 2.7-A crystal structure of intracellular antibody folds
based on scaffolds developed from intracellular antibody capture technology, and
we reveal that there is no structural or functional difference with or without
the intra-domain disulfide bond of the variable domain of heavy chain or the
variable domain of light chain. The data indicate that, in the reducing in vivo
environment, the absence of the intra-domain disulfide bond is not an impediment
to correction of antibody folding or to interaction with antigen. Thus, the
structural constraints for in-cell function are intrinsic to variable
single-domain framework sequences, providing a generic scaffold for isolation of
functional intracellular antibody single domains.
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Figure 3.
Fig. 3. Structural comparison of native and disulfide-free
RAS–anti-RAS Fv complex. (a) Representation of HRAS bound by
disulfide-free Fv. HRAS(G12V) (green) is shown as a molecular
surface model. The switch I and switch II regions of RAS are
shown in cyan and magenta, respectively, and guanidine
triphosphate (GTP) is shown in orange. The Fv [comprising VH
(blue) and VL (red)] is shown as a ribbon representation, with
the CDRs of VH and VL in blue and pink, respectively. The
positions 23 and 104 where cysteines were substituted by
alanine and valine in VH, or by valine and alanine in VL, are in
yellow. (b) Superimposition of anti-RAS Fv native form^2 and
disulfide-free Fv with alanine–valine substitutions in VH
[VH#6(AV)] and with valine–alanine substitutions in VL(VA).
The structure is shown as a stereo view of the Cα trace VH and
VL of the native forms shown in cyan and pink and of the
disulfide-free forms shown in blue and red, respectively. (c and
d) 2F[o] − F[c] electron density maps (contoured at 0.5σ)
around the disulfide bond regions of VH (c) and VL (d): native
form (left) and disulfide-free mutant (right). The distance of
Cα atom between the residues of cysteines or their
substitutions is shown as a dotted line in yellow.
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Figure 4.
Fig. 4. Electron density maps of VH and VL CDRs. Views of VH
and VL CDR structures with a 2F[o] − F[c] electron density
map. The map is contoured at 0.5σ. Left panels show the native
anti-RAS VH (a–c) and VL (d–f) single domains. Right panels
show the disulfide-free form. (a) VHCDR1; (b) VHCDR2; (c)
VHCDR3; (d) VLCDR1; (e) VLCDR2; (f) VLCDR3.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
376,
749-757)
copyright 2008.
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Secondary reference #1
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Title
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Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with ras.
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Authors
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T.Tanaka,
R.L.Williams,
T.H.Rabbitts.
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Ref.
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EMBO J, 2007,
26,
3250-3259.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4 Crystal structure of the RAS–anti-RAS single domains
complex. HRAS(G12V) protein complexed with the anti-RAS #6 in an
Fv format is shown in ribbon form (A) or space filling (B),
where HRAS(G12V) is shown in green and the Fv proteins VH and VL
are shown in cyan and orange, respectively. The CDRs of VH and
VL are in yellow and lemon and the RAS switch I and II regions
are in red and purple, respectively. The GTP and Mg^2+ ion in
RAS are in blue and magenta, respectively.
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Figure 5.
Figure 5 The binding site of the single domains on the RAS
molecule. (A) A stereo diagram of the HRAS(G12V)-GTP-Fv binding
interface. HRAS(G12V) is in green and the VH and VL chains are
in cyan and orange, respectively. The CDRs of VH and VL are in
yellow and lemon and the RAS switch I and II regions are in red
and purple, respectively. Residues involved in the interface are
shown in cylinder configuration. Specific residues of RAS are
shown in blue, VH in red and VL in brown. Putative hydrogen
bonds are indicated by dashed lines. (B) Schematic
representation of the interacting residues in HRAS (green) and
in the anti-RAS antibody (VH, yellow; VL, lemon). Putative
hydrogen bonds are indicated by dotted lines. (C) The structures
of HRAS(G12V)-GTP (green, red and purple) bound to anti-RAS Fv
and of HRAS-GDP (blue) (PDB, 4Q21) (Milburn et al, 1990) are
superimposed to illustrate the selectivity of iDab#6 single VH
domain binding to activated GTP-bound RAS.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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