PDBsum entry 3ch8

Structural protein

PDB id: 3ch8

Contents

Protein chain

190 a.a. *

Ligands

PRO-GLN-PRO-VAL-ASP-SER-TRP-VAL

Metals

_MG

Waters ×89

* Residue conservation analysis

PDB id:

3ch8

Name:

Structural protein

Title:

The crystal structure of pdz-fibronectin fusion protein

Structure:

Fusion protein pdz-fibronectin,fibronectin. Chain: a. Synonym: fn,cold-insoluble globulin,cig. Engineered: yes. Other_details: see remark 400,see remark 400. C-terminal octapeptide from protein arvcf. Chain: p. Fragment: c-terminal residues. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: fn1, fn. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes. Synthetic construct. Organism_taxid: 32630

Resolution:

1.90Å R-factor: 0.204 R-free: 0.260

Authors:

K.Makabe,J.Huang,A.Koide,S.Koide

Key ref:

J.Huang et al. (2009). Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution. J Mol Biol, 392, 1221-1231. PubMed id: 19646997

Date:

08-Mar-08 Release date: 31-Mar-09

Protein chain

P02751  (FINC_HUMAN) -  Fibronectin from Homo sapiens

Seq: 2477 a.a.

Struc: 190 a.a.*

* PDB and UniProt seqs differ at 104 residue positions (black crosses)

J Mol Biol 392:1221-1231 (2009)

PubMed id: 19646997

Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution.

J.Huang, K.Makabe, M.Biancalana, A.Koide, S.Koide.

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.