9DB5

A DARPin fused to the 1TEL crystallization chaperone via a proline-alanine linker

9DB5 の概要

• エントリーDOI: 10.2210/pdb9db5/pdb
• 分子名称: Transcription factor ETV6,DARPin, ACETIC ACID, SODIUM ION, ... (5 entities in total)
• 機能のキーワード: telsam, darpin, etv6, designed ankyrin repeat protein, protein binding
• 由来する生物種: Homo sapiens (human); 詳細
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 26779.88

構造登録者

Pedroza Romo, M.J.,Averett, J.C.,Keliiliki, A.,Wilson, E.W.,Smith, C.,Hansen, D.,Averett, B.,Gonzalez, J.,Noakes, E.,Nickles, R.,Doukov, T.,Moody, J.D. (登録日: 2024-08-23, 公開日: 2024-10-09, 最終更新日: 2025-10-01)

主引用文献

Pedroza Romo, M.J.,Keliiliki, A.,Averett, J.C.,Gonzalez, J.F.,Noakes, E.,Wilson, E.W.,Smith, C.,Averett, B.,Hansen, D.,Nickles, R.,Bradford, M.,Soleimani, S.,Smith, T.,Nawarathnage, S.,Samarwickrama, P.,Kelsch, A.,Bunn, D.,Stewart, C.,Abiodun, W.,Tsubaki, E.,Brown, S.,Doukov, T.I.,Moody, J.D.
Optimal TELSAM-Target Protein Linker Character is Target Protein-Dependent.
Biorxiv, 2025

PubMed Abstract: Fusing a variant of the sterile alpha motif domain of the human translocation ETS leukaemia protein (TELSAM) to a protein of interest has been shown to significantly enhance crystallization propensity. TELSAM is a pH-dependent, polymer-forming protein crystallization chaperone which, when covalently fused to a protein of interest, forms a stable, well-ordered crystal lattice. However, despite its success, a challenge persists in that crystal quality and diffraction limits appear to be heavily dependent on the choice of linker between TELSAM and the protein of interest, with identification of a functional linker relying on trial-and-error methods. Likewise, previous studies revealed that the 10xHis tag at the TELSAM N-terminus can either facilitate or hinder the ordered crystallization of target proteins attached via flexible or semi-flexible linkers. To address these challenges, we designed multiple constructs with several types of linkers-rigid (helical fusion), semi-flexible (Pro-Ala), and flexible (poly-Gly)-of varying lengths to fuse a designed ankyrin repeat protein (DARPin) to the TELSAM C-terminus. Semi-flexible and flexible linker constructs were made with and without the 10xHis tag. Our findings indicate that short semi-flexible and rigid linkers consistently yield large crystals within 24 hours with a DARPin target protein, but that flexible linkers perform best with a TNK1 UBA domain target protein. Removing the 10xHis tag enhanced crystallization rates, improved crystal morphology, and increased the crystallization propensity of semi-flexible and flexible linker constructs. While removing the His tag did not have a significant effect on crystal size, it improved the diffraction limits and crystal quality of the 1TEL-PA-DARPin construct. These results suggest that the ideal linker selection primarily depends on the properties of the target protein. Our data support the recommendation to use a short yet flexible or semi-flexible linker between TELSAM and the target protein to facilitate protein crystallization and high-resolution structure determination.

PubMed: 40950120
DOI: 10.1101/2025.08.29.672704

実験手法

X-RAY DIFFRACTION (1.57 Å)