PDBsum entry 2qyj

De novo protein

PDB id: 2qyj

Contents

Protein chain

154 a.a. *

Ligands

SO4 ×4

Waters ×111

* Residue conservation analysis

PDB id:

2qyj

Name:

De novo protein

Title:

Crystal structure of a designed full consensus ankyrin

Structure:

Ankyrin ni3c. Chain: a. Engineered: yes

Source:

Synthetic: yes. Other_details: the peptide was chemically synthesized.

Resolution:

2.05Å R-factor: 0.188 R-free: 0.226

Authors:

T.Merz

Key ref:

T.Merz et al. (2008). Stabilizing ionic interactions in a full-consensus ankyrin repeat protein. J Mol Biol, 376, 232-240. PubMed id: 18155045 DOI: 10.1016/j.jmb.2007.11.047

Date:

15-Aug-07 Release date: 13-Nov-07

Protein chain

No UniProt id for this chain

Struc: 154 a.a.

DOI no: 10.1016/j.jmb.2007.11.047

J Mol Biol 376:232-240 (2008)

PubMed id: 18155045

Stabilizing ionic interactions in a full-consensus ankyrin repeat protein.

T.Merz, S.K.Wetzel, S.Firbank, A.Plückthun, M.G.Grütter, P.R.Mittl.

ABSTRACT

Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 A resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI(3)C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.

Selected figure(s)

Figure 1.
Fig. 1. (a) Ribbon diagram of the NI[3]C structure. The terminal and internal repeats are in magenta and green, respectively. (b) Stereo view of the interactions involving the three sulfate ions and the randomized residues. Dashed lines in black denote HB between lysine and aspartate residues at positions 2 and 3, as well as HB between arginine and glutamate residues at positions 13 and 14. Dashed lines in blue denote all interactions of the sulfate ions.

Figure 4.
Fig. 4. Electrostatic surface potentials, calculated using the Adaptive Poisson–Boltzmann Solver^21 of (a) 4ANK (PDB code 1N0R) and (b) NI[3]C. Both representations show the concave binding site with the N- and C-termini on the right and left sides, respectively. Bound sulfate ions in the NI[3]C molecule are shown as spheres. Both surfaces were in blue and red for positive and negative electrostatic charge densities (same scale), respectively.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 376, 232-240) copyright 2008.

Figures were selected by an automated process.