8CPN

Crystal structure of the PolB16_OarG intein variant S1A, N183A

8CPN の概要

• エントリーDOI: 10.2210/pdb8cpn/pdb
• 分子名称: PolB16 intein, IODIDE ION (3 entities in total)
• 機能のキーワード: split intein, ligation, protein engineering, splicing
• 由来する生物種: metagenome
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 25274.26

構造登録者

Kattelmann, S.,Pasch, T.,Mootz, H.D.,Kummel, D. (登録日: 2023-03-03, 公開日: 2023-05-17, 最終更新日: 2024-06-19)

主引用文献

Pasch, T.,Schroder, A.,Kattelmann, S.,Eisenstein, M.,Pietrokovski, S.,Kummel, D.,Mootz, H.D.
Structural and biochemical analysis of a novel atypically split intein reveals a conserved histidine specific to cysteine-less inteins.
Chem Sci, 14:5204-5213, 2023

PubMed Abstract: Protein -splicing mediated by a split intein reconstitutes a protein backbone from two parts. This virtually traceless autoprocessive reaction provides the basis for numerous protein engineering applications. Protein splicing typically proceeds through two thioester or oxyester intermediates involving the side chains of cysteine or serine/threonine residues. A cysteine-less split intein has recently attracted particular interest as it can splice under oxidizing conditions and is orthogonal to disulfide or thiol bioconjugation chemistries. Here, we report the split PolB16 OarG intein, a second such cysteine-independent intein. As a unique trait, it is atypically split with a short intein-N precursor fragment of only 15 amino acids, the shortest characterized to date, which was chemically synthesized to enable protein semi-synthesis. By rational engineering we obtained a high-yielding, improved split intein mutant. Structural and mutational analysis revealed the dispensability of the usually crucial conserved motif N3 (block B) histidine as an obvious peculiar property. Unexpectedly, we identified a previously unnoticed histidine in hydrogen-bond forming distance to the catalytic serine 1 as critical for splicing. This histidine has been overlooked so far in multiple sequence alignments and is highly conserved only in cysteine-independent inteins as a part of a newly discovered motif NX. The motif NX histidine is thus likely of general importance to the specialized environment in the active site required in this intein subgroup. Together, our study advances the toolbox as well as the structural and mechanistic understanding of cysteine-less inteins.

PubMed: 37206380
DOI: 10.1039/d3sc01200j

実験手法

X-RAY DIFFRACTION (1.85 Å)