 |
PDBsum entry 3ch8
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Structural protein
|
PDB id
|
|
|
|
3ch8
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structural basis for exquisite specificity of affinity clamps, Synthetic binding proteins generated through directed domain-Interface evolution.
|
|
|
Authors
|
|
J.Huang,
K.Makabe,
M.Biancalana,
A.Koide,
S.Koide.
|
|
|
Ref.
|
|
J Mol Biol, 2009,
392,
1221-1231.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
We have established a new protein-engineering strategy termed "directed
domain-interface evolution" that generates a binding site by linking two
protein domains and then optimizing the interface between them. Using this
strategy, we have generated synthetic two-domain "affinity clamps"
using PDZ and fibronectin type III (FN3) domains as the building blocks. While
these affinity clamps all had significantly higher affinity toward a target
peptide than the underlying PDZ domain, two distinct types of affinity clamps
were found in terms of target specificity. One type conserved the specificity of
the parent PDZ domain, and the other increased the specificity dramatically.
Here, we characterized their specificity profiles using peptide phage-display
libraries and scanning mutagenesis, which suggested a significantly enlarged
recognition site of the high-specificity affinity clamps. The crystal structure
of a high-specificity affinity clamp showed extensive contacts with a portion of
the peptide ligand that is not recognized by the parent PDZ domain, thus
rationalizing the improvement of the specificity of the affinity clamp. A
comparison with another affinity clamp structure showed that, although both had
extensive contacts between PDZ and FN3 domains, they exhibited a large offset in
the relative position of the two domains. Our results indicate that linked
domains could rapidly fuse and evolve as a single functional module, and that
the inherent plasticity of domain interfaces allows for the generation of
diverse active-site topography. These attributes of directed domain-interface
evolution provide facile means to generate synthetic proteins with a broad range
of functions.
|
|
|
|
|
|
|
