 |
PDBsum entry 2cbq
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Antimicrobial
|
PDB id
|
|
|
|
2cbq
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways.
|
|
|
Authors
|
|
A.Drevelle,
M.Graille,
B.Heyd,
I.Sorel,
N.Ulryck,
F.Pecorari,
M.Desmadril,
H.Van tilbeurgh,
P.Minard.
|
|
|
Ref.
|
|
J Mol Biol, 2006,
358,
455-471.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
We have recently applied in vitro evolution methods to create in
Neocarzinostatin a new binding site for a target molecule unrelated to its
natural ligand. The main objective of this work was to solve the structure of
some of the selected binders in complex with the target molecule: testosterone.
Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of
sequence families that came out of the selection process within different
randomization schemes. In order to evaluate ligand-induced conformational
adaptation, we also determined the structure of one of the proteins (3.24) in
the free and complexed forms. Surprisingly, all these mutants bind not one but
two molecules of testosterone in two very different ways. The 3.24 structure
revealed that the protein spontaneously evolved in the system to bind two ligand
molecules in one single binding crevice. These two binding sites are formed by
substituted as well as by non-variable side-chains. The comparison with the free
structure shows that only limited structural changes are observed upon ligand
binding. The X-ray structures of the complex formed by 1a.15 and 4.1
Neocarzinostatin mutants revealed that the two variants form very similar
dimers. These dimers were observed neither for the uncomplexed variants nor for
wild-type Neocarzinostatin but were shown here to be induced by ligand binding.
Comparison of the three complexed forms clearly suggests that these
unanticipated structural responses resulted from the molecular arrangement used
for the selection experiments.
|
|
|
|
|
|
|
|
Figure 8.
Figure 8. Schematic representation of the evolutionary
pathway. Positions randomized in the three libraries are
indicated in green, those randomized only in library 4 are in
purple. NNK and RVY refer to the degenerated nucleotide sequence
used to randomize the indicated amino acid during library
construction (NNK code for 20 amino acids, RVY code
for:A,S,T,N,D,G). The more constrained positions 35 and 96 are
indicated in red and cyan. THS molecules are in blue.
|
|
Figure 9.
Figure 9. Model of the ternary complex
streptavin/testobiotin–3.24 variant. The experimental
structure of 3.24 variant complexed with THS and the
biotin-bound streptavidin tetramer (PDB code: 1STP) were used to
build this model. The model shows two streptavidin monomers
displaying biotinylated testosterone (orange) on the same face
of streptavidin tetramer (yellow/orange). The 3.24 variant is in
purple with the bound biotinylated testosterone molecules in
dark grey. This model suggest that the concomitant binding of
two streptavidin-bound testosterone molecules is compatible with
the linker length and known structures of the 3.24 variant and
streptavidin-binding sites.
|
|
|
|
|
|
The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
358,
455-471)
copyright 2006.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
In vitro evolution of the binding specificity of neocarzinostatin, An enediyne-Binding chromoprotein.
|
|
|
Authors
|
|
B.Heyd,
F.Pecorari,
B.Collinet,
E.Adjadj,
M.Desmadril,
P.Minard.
|
|
|
Ref.
|
|
Biochemistry, 2003,
42,
5674-5683.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
