PDBsum entry 1mj0

De novo protein

PDB id: 1mj0

Contents

Protein chain

156 a.a. *

Ligands

SO4 ×2

TRS

Waters ×178

* Residue conservation analysis

PDB id:

1mj0

Name:

De novo protein

Title:

Sank e3_5: an artificial ankyrin repeat protein

Structure:

Sank e3_5 protein. Chain: a. Engineered: yes

Source:

Designed synthetic gene. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.03Å R-factor: 0.184 R-free: 0.230

Authors:

A.Kohl,H.K.Binz,P.Forrer,M.T.Stumpp,A.Plueckthun,M.G.Gruetter

Key ref:

A.Kohl et al. (2003). Designed to be stable: crystal structure of a consensus ankyrin repeat protein. Proc Natl Acad Sci U S A, 100, 1700-1705. PubMed id: 12566564 DOI: 10.1073/pnas.0337680100

Date:

26-Aug-02 Release date: 21-Jan-03

Protein chain

No UniProt id for this chain

Struc: 156 a.a.

DOI no: 10.1073/pnas.0337680100

Proc Natl Acad Sci U S A 100:1700-1705 (2003)

PubMed id: 12566564

Designed to be stable: crystal structure of a consensus ankyrin repeat protein.

A.Kohl, H.K.Binz, P.Forrer, M.T.Stumpp, A.Plückthun, M.G.Grütter.

ABSTRACT

Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-A resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the beta-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

Selected figure(s)

Figure 2.
Fig. 2. Structure of the consensus AR protein E3_5. (A and B) Perpendicular views of E3_5 prepared with MOLMOL (53). Ribbon representation of E3_5 showing the helices of the N-terminal, internal (consensus), and C-terminal repeats in green, dark blue, and light blue, respectively. The side chains of amino acids at randomized positions are highlighted in red. (C) The consensus AR sequence. X, any amino acid but C, G, or P; Z, any of the amino acids H, N, Y.

Figure 3.
Fig. 3. H-bonding networks in the consensus AR protein E3_5. H-bonds are shown in green. (A) -Turn H-bond network spanning the entire molecule. Each -turn is formed by the sequence X[33]Asp[1]X[2]X[3]Gly[4]X[5] (X[i] being randomized positions). The X[2] positions in the first three -turns are labeled for orientation (Asn-45, Leu-78, and Asn-111). (B) TPLH motifs of repeats 2 and 3. The side chains of His-52 and His-85 form H-bonds to the -turn of the position X[5] of the third or fourth repeat, respectively.

Figures were selected by the author.