7CFV

Solution NMR structure of DnaX mini intein from Spirulina platensis

7CFV の概要

• エントリーDOI: 10.2210/pdb7cfv/pdb
• 分子名称: Spl DnaX mini-intein (1 entity in total)
• 機能のキーワード: spl dnax, intein, protein splicing, splicing
• 由来する生物種: Arthrospira platensis C1
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 15809.87

構造登録者

Boral, S.,Lee, W.,De, S. (登録日: 2020-06-28, 公開日: 2020-12-23, 最終更新日: 2024-05-15)

主引用文献

Boral, S.,Maiti, S.,Basak, A.J.,Lee, W.,De, S.
Structural, Dynamic, and Functional Characterization of a DnaX Mini-intein Derived from Spirulina platensis Provides Important Insights into Intein-Mediated Catalysis of Protein Splicing.
Biochemistry, 59:4711-4724, 2020

PubMed Abstract: Protein splicing is a self-catalyzed post-translational modification in which the intein enzyme excises itself from a precursor protein and ligates the flanking sequences to produce a mature protein. We report the solution structure of a 136-residue DnaX mini-intein enzyme derived from the cyanobacterium . This sequence adopts a well-defined globular structure and forms a horseshoe-shaped fold commonly found in the HINT (hedgehog intein) topology. Backbone dynamics and hydrogen exchange experiments revealed conserved motions on various time scales, which is proposed to be a characteristic of the intein fold. Interestingly, several dynamic motions were found in symmetrically equivalent positions within the protein structure, which might be a consequence of the symmetrical intein fold. In cell splicing activity showed that DnaX mini-intein is a highly active enzyme. The precursor protein was not detected at any timepoint of the assay. Apart from the splicing reaction, catalytic cleavage at the N- and C-termini of the precursor protein was also observed. To determine the roles of the catalytic residues in splicing and cleavage reactions, all combinations of alanine mutations of these residues were generated and functionally characterized. This in-depth analysis revealed cooperativity between these catalytic residues, which suppresses the N- and C-terminal cleavage reactions and enhances the yield of the spliced product. Overall, this study provides a thorough structural, dynamic, and functional characterization of a new intein sequence and adds to the collection of these unique enzymes that have found tremendous applications in biochemistry and biotechnology.

PubMed: 33289560
DOI: 10.1021/acs.biochem.0c00828

実験手法

SOLUTION NMR