8AES

Crystal structure of a thermophilic O6-alkylguanine-DNA alkyltransferase-derived self-labeling protein-tag

8AES の概要

• エントリーDOI: 10.2210/pdb8aes/pdb
• 分子名称: Methylated-DNA--protein-cysteine methyltransferase (2 entities in total)
• 機能のキーワード: self-labelling protein tag, rational mutagenesis, o6-alkylguanine-dna alkyltransferase, clip-tag., transferase
• 由来する生物種: Saccharolobus solfataricus
• タンパク質・核酸の鎖数: 10
• 化学式量合計: 190491.76

構造登録者

Genta, M.,Perugino, G.,Miggiano, R. (登録日: 2022-07-13, 公開日: 2022-10-12, 最終更新日: 2024-11-20)

主引用文献

Merlo, R.,Mattossovich, R.,Genta, M.,Valenti, A.,Di Mauro, G.,Minassi, A.,Miggiano, R.,Perugino, G.
First thermostable CLIP- tag by rational design applied to an archaeal O 6 -alkyl-guanine-DNA-alkyl-transferase.
Comput Struct Biotechnol J, 20:5275-5286, 2022

PubMed Abstract: Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-, CLIP- and ). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon (OGT), with no DNA-binding activity, able to covalently react with -benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-, named OGT- . The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from OGT, led to the production of OGT- was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP- establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any -alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP- variant.

PubMed: 36212535
DOI: 10.1016/j.csbj.2022.09.015

実験手法

X-RAY DIFFRACTION (2.8 Å)