An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

Abstract

Background: Intein-mediated protein splicing involves rearrangement of the polypeptide backbone through ester intermediates and excises the intein from the precursor. Results: An unusual split intein was identified that converts an oxoester into a thioester. Conclusion: Inteins can catalyze two consecutive formally uphill reactions. Significance: The first example of this special case in protein splicing underlines the mechanistic diversity of inteins.

N‐terminal chemical protein labeling using the naturally split GOS‐TerL intein

“…A unique feature of the GOS‐TerL intein is the Ser1 instead of a Cys1 residue at the splice junction in the Int N fragment . Because also the remainder of the Int N sequence lacks any cysteine residues, we decided to explore our previously reported CysTag technology using the GOS‐TerL intein.…”

“…The final construct CysTag‐Int N ‐MBP (protein 1 ), with an MGCH 8 MEFE sequence as the N‐terminal CysTag sequence, is illustrated in Figure (A). To test the tolerance of the intein towards the large MBP, intended to be excised together with the Int N fragment during the splicing reaction, purified 1 was incubated with Int C ‐Trx‐H 6 (protein 2 ) , containing thioredoxin (Trx) as the model protein of interest (Figure (B)). Figure (C) shows that nearly quantitative turnover of the intein constructs was achieved within 15 min with formation of the desired splice product MGCH 8 MEFE‐Trx‐H 6 ( 3 ) and the remaining intein proteins Int N ‐MBP ( 4 ) and Int C ( 5 ).…”

“…The encoded sequence is as follows: MGCHHHHHHHHMEFE‐Int N (1–28)‐GSGSGSSR‐MBP. The previously described pAB05 encodes Int C ‐Trx‐H 6 ( 2 ) with His‐patch thioredoxin from Escherichia coli in a pBAD202 vector backbone . Four native amino acids (CEFLG) were kept C‐terminal to the Int C fragment.…”

“…We have recently reported the biochemical characterization of the Global Ocean Sampling Project and Terminase Large Subunit (GOS‐TerL) intein, which was identified from metagenomic DNA databases . It represents a naturally split intein with Int N and Int C fragment sizes of 37 and 115 aa, respectively.…”

“…The second highly unusual feature of the GOS‐TerL intein is the combination of the amino acids at the positions 1 and +1 downstream of the N‐terminal and C‐terminal scissile bonds, respectively, between intein and extein fragments. The GOS‐TerL intein employs the unique combination of Ser1 and Cys + 1 residues that suggests an acyl shift from the linear ester intermediate to a branched thioester intermediate. With these properties, the Int N fragment of the GOS‐TerL intein would also lend itself suitable to the CysTag approach.…”