2JAB

A designed ankyrin repeat protein evolved to picomolar affinity to Her2

2JAB の概要

• エントリーDOI: 10.2210/pdb2jab/pdb
• 分子名称: H10-2-G3 (2 entities in total)
• 機能のキーワード: her2, darpin, ankyrin repeat protein, membrane protein, human epidermal growth factor receptor 2, de novo protein
• 由来する生物種: SYNTHETIC CONSTRUCT
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 43855.31

構造登録者

Zahnd, C.,Wyler, E.,Schwenk, J.M.,Steiner, D.,Lawrence, M.C.,McKern, N.M.,Pecorari, F.,Ward, C.W.,Joos, T.O.,Pluckthun, A. (登録日: 2006-11-27, 公開日: 2007-05-08, 最終更新日: 2023-12-13)

主引用文献

Zahnd, C.,Wyler, E.,Schwenk, J.M.,Steiner, D.,Lawrence, M.C.,Mckern, N.M.,Pecorari, F.,Ward, C.W.,Joos, T.O.,Pluckthun, A.
A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2
J.Mol.Biol., 369:1015-, 2007

PubMed Abstract: Designed ankyrin repeat proteins (DARPins) are a novel class of binding molecules, which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (average 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma extracts. We have determined the crystal structure of this DARPin at 1.7 A, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An analysis of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.

PubMed: 17466328
DOI: 10.1016/J.JMB.2007.03.028

実験手法

X-RAY DIFFRACTION (1.7 Å)