PDBsum entry 4jw2

De novo protein/protein binding

PDB id: 4jw2

Contents

Protein chains

73 a.a.

190 a.a.

Ligands

EDO ×3

Waters ×146

PDB id:

4jw2

Name:

De novo protein/protein binding

Title:

Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alpharep)

Structure:

A3 artificial protein. Chain: a. Engineered: yes. Ba3-2: binder of a3 protein. Chain: b. Engineered: yes

Source:

Synthetic construct. Artificial. Organism_taxid: 32630. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.90Å R-factor: 0.195 R-free: 0.224

Authors:

A.Guellouz,M.Valerio-Lepiniec,A.Urvoas,A.Chevrel,M.Graille,Z.Fourati- Kammoun,M.Desmadril,H.Van Tilbeurgh,P.Minard

Key ref:

A.Guellouz et al. (2013). Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep). Plos One, 8, e71512. PubMed id: 24014183 DOI: 10.1371/journal.pone.0071512

Date:

27-Mar-13 Release date: 25-Sep-13

Protein chain

No UniProt id for this chain

Struc: 73 a.a.

Protein chain

No UniProt id for this chain

Struc: 190 a.a.

DOI no: 10.1371/journal.pone.0071512

Plos One 8:e71512 (2013)

PubMed id: 24014183

Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep).

A.Guellouz, M.Valerio-Lepiniec, A.Urvoas, A.Chevrel, M.Graille, Z.Fourati-Kammoun, M.Desmadril, H.van Tilbeurgh, P.Minard.

ABSTRACT

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.