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PDBsum entry 1n0s
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Binding protein
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PDB id
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1n0s
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Contents |
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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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Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region.
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Authors
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I.P.Korndörfer,
G.Beste,
A.Skerra.
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Ref.
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Proteins, 2003,
53,
121-129.
[DOI no: ]
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PubMed id
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Abstract
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The artificial lipocalin FluA with novel specificity toward fluorescein was
derived via combinatorial engineering from the bilin-binding protein, BBP by
exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal
structure of FluA at 2.0 A resolution in the space group P2(1) with two
protein-ligand complexes in the asymmetric unit. In both molecules, the
characteristic beta-barrel architecture with the attached alpha-helix is well
preserved. In contrast, the four loops at one end of the beta-barrel that form
the entrance to the binding site exhibit large conformational deviations from
the wild-type protein, which can be attributed to the sidechain replacements.
Specificity for the new ligand is furnished by hydrophobic packing, charged
sidechain environment, and hydrogen bonds with its hydroxyl groups.
Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin
IX(gamma) in the natural lipocalin. Triggered by the substituted residues,
unmutated sidechains at the bottom of the binding site adopt conformations that
are quite different from those observed in the BBP, illustrating that not only
the loop region but also the hydrophobic interior of the beta-barrel can be
reshaped for molecular recognition. Particularly, Trp 129 participates in a
tight stacking interaction with the xanthenolone moiety, which may explain the
ultrafast electron transfer that occurs on light excitation of the bound
fluorescein. These structural findings support our concept of using lipocalins
as a scaffold for the engineering of so-called "anticalins" directed
against prescribed targets as an alternative to recombinant antibody fragments.
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Figure 2.
Figure 2. The two monomers of FluA in the asymmetric crystal
unit. Each polypeptide chain is shown in a ribbon presentation,
light- and dark-gray, respectively, with its N- and C-terminus
labeled and the fluorescein ligand depicted as a ball-and-stick
model. The intermolecular  -sheet
that is formed via association of the loop #3 segments from the
two monomers can be seen at the middle, together with the
position of the crystallographically fixed sulfate ion.
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Figure 4.
Figure 4. Stereo images with details of the novel
ligand-binding site in FluA. (A) 2F[o]-F[c] electron density for
the bound fluorescein contoured at 1.0  ,
together with sidechains of residues closer than 3.8 Å.
Amino acids that were mutated in FluA (cf. Fig. 1) are colored
dark-green, whereas original residues of BBP are depicted in
light-green. (B) The set of 16 randomly mutated sidechains in
the FluA crystal structure with the complexed fluorescein. (C)
Arrangement of positively charged residues at the entrance to
the ligand pocket of FluA. The three characteristically arranged
Arg side chains (see text) are colored dark-blue. Note that
there is a considerable distance between Arg 95 - whose
sidechain is well defined in the electron density - and the
bound fluorescein along the view axis (distance between the
guanidinium and carboxylate groups: 8.6 Å) such that no
direct contact is formed.
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The above figures are
reprinted
by permission from John Wiley & Sons, Inc.:
Proteins
(2003,
53,
121-129)
copyright 2003.
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