The crystal structure of the naturally split gp41‐1 intein guides the engineering of orthogonal split inteins from cis‐splicing inteins

• Published in: The FEBS journal Vol. 287; no. 9; pp. 1886 - 1898
• Main Authors: Beyer, Hannes M., Mikula, Kornelia M., Li, Mi, Wlodawer, Alexander, Iwaï, Hideo
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-05-2020
• Subjects: Crystal structure; Crystallography, X-Ray; Glycoprotein gp41; gp41‐1 intein; Inteins; Metagenomics; Models, Molecular; orthogonal split intein; Protein Engineering; Protein Splicing; Proteins; Splicing; Stereoisomerism; crystal structure; protein engineering; orthogonal split intein; protein splicing; gp41-1 intein
• ISSN: 1742-464X; 1742-4658; 1742-4658
• DOI: 10.1111/febs.15113

Protein trans‐splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Å‐resolution crystal structure of a fused variant from the naturally split gp41‐1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125‐residue gp41‐1 intein revealed a compact pseudo‐C2‐symmetry commonly found in the Hedgehog/Intein superfamily with extensive charge–charge interactions between the split N‐ and C‐terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis‐splicing intein. This strategy could be applicable for creating novel natural‐like split inteins from other, more prevalent cis‐splicing inteins.
Database
Structural data are available in the RCSB Protein Data Bank under the accession number 6QAZ.
The high‐resolution crystal structure of the naturally split gp41‐1 intein revealed an intense charge network between the intein split fragments. We grafted a similar charge network onto the cis‐splicing NpuDnaB mini‐intein to design artificial natural‐like split intein variants with orthogonal character.