4JW3

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

4JW3 の概要

• エントリーDOI: 10.2210/pdb4jw3/pdb
• 関連するPDBエントリー: 3LTM 4JW2
• 分子名称: Neocarzinostatin, Alpha-helical artificial proteins (3 entities in total)
• 機能のキーワード: alpha-helical protein, protein engineered to interact with protein of interest, ncs 3tes24 variant, dna binding protein-de novo protein complex, dna binding protein/de novo protein
• 由来する生物種: Streptomyces malayensis; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 55379.60

構造登録者

Guellouz, A.,Valerio-Lepiniec, M.,Urvoas, A.,Chevrel, A.,Graille, M.,Fourati-Kammoun, Z.,Desmadril, M.,van Tilbeurgh, H.,Minard, P. (登録日: 2013-03-27, 公開日: 2013-09-25, 最終更新日: 2024-10-09)

主引用文献

Guellouz, A.,Valerio-Lepiniec, M.,Urvoas, A.,Chevrel, A.,Graille, M.,Fourati-Kammoun, Z.,Desmadril, M.,van Tilbeurgh, H.,Minard, P.
Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep).
Plos One, 8:e71512-e71512, 2013

PubMed 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.

PubMed: 24014183
DOI: 10.1371/journal.pone.0071512

実験手法

X-RAY DIFFRACTION (2.6 Å)