PDBsum entry 2qbw

Unknown function

PDB id: 2qbw

Contents

Protein chain

189 a.a. *

Ligands

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

Waters ×214

* Residue conservation analysis

PDB id:

2qbw

Name:

Unknown function

Title:

The crystal structure of pdz-fibronectin fusion protein

Structure:

Pdz-fibronectin fusion protein. Chain: a. Synonym: erbb2-interacting protein, erbin, densin-180-like protein. Engineered: yes. Polypeptide. Chain: b. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: erbb2ip. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Other_details: chemically synthesized

Resolution:

1.80Å R-factor: 0.170 R-free: 0.205

Authors:

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

Key ref:

J.Huang et al. (2008). Design of protein function leaps by directed domain interface evolution. Proc Natl Acad Sci U S A, 105, 6578-6583. PubMed id: 18445649 DOI: 10.1073/pnas.0801097105

Date:

18-Jun-07 Release date: 22-Apr-08

Protein chain

Q96RT1  (ERBIN_HUMAN) -  Erbin from Homo sapiens

Seq: 1412 a.a.

Struc: 189 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1073/pnas.0801097105

Proc Natl Acad Sci U S A 105:6578-6583 (2008)

PubMed id: 18445649

Design of protein function leaps by directed domain interface evolution.

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

ABSTRACT

Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting "affinity clamp" had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.

Selected figure(s)

Figure 1.
The concept of directed domain interface evolution and building blocks used in this work. (A and B) Comparison of domain interface engineering with conventional protein engineering. In the conventional engineering that mimics gene duplication and sequence divergence (A), the interface predefined in the starting scaffold is altered/refined, which tends to produce incremental changes in function. In contrast, domain interface engineering that mimics gene combination and sequence divergence (B) produces a new functional site at the interface between two domains, which can result in a major leap in protein function. (C) The structure of the Erbin PDZ bound to a peptide (PDB entry 1MFG). The N and C termini are indicated. The positions for the new termini of the circularly permutated PDZ (cpPDZ) are shown with a triangle and residue numbers. Right shows the surface of the PDZ domain with the peptide as a stick model, illustrating the shallow binding pocket. (D) The structure of FN3 (PDB entry 1FNF). The loops that are diversified to construct combinatorial libraries are labeled. The termini are also labeled. Note that the N terminus and the recognition loops are located on the same side of the FN3 protein.

Figure 3.
The x-ray crystal structure of ePDZ-a in complex with the ARVCF peptide. (A) Ribbon representations of the overall structure. The cpPDZ and FN3 portions and the peptide are shown in gray, cyan, and yellow, respectively. Missing residues for the linker segment are indicated with dashed lines. (B) Clamping of the peptide by ePDZ-a. Only the region in the dashed box in A is shown. The surfaces originating from the cpPDZ and FN3 portions are shown in gray and cyan, respectively, and the peptide is shown as a yellow stick model. (C) Interactions of the FN3 loops with the cpPDZ/peptide complex. The three FN3 loops (BC, DE, and FG loops) are shown as sticks in blue, cyan, and red, respectively. The surface of the PDZ portion is shown in gray, and the peptide is shown as yellow spheres. In Lower, the surfaces of the PDZ and peptide portions in contact with the BC, DE, and FG loops (within 5 Å are shown in blue, cyan, and red, respectively, and those in contact with both BC and FG loops are in magenta. The peptide surfaces without FN3 contact are shown in yellow, and the green line encloses the bound peptide. (D) Superposition of wild-type PDZ (PDB entry 1MFG, green) and cpPDZ (gray). The original and new termini are indicated. The rmsd for the equivalent 97 Cα atoms was 0.54 Å. The structures are in the same orientation as in A.

Figures were selected by the author.