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A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine

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Abstract

Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.

Graphical abstract: A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine

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Publication details

The article was received on 06 Mar 2018, accepted on 03 Oct 2018 and first published on 03 Oct 2018


Article type: Edge Article
DOI: 10.1039/C8SC01074A
Citation: Chem. Sci., 2019, Advance Article
  • Open access: Creative Commons BY license
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    A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine

    K. Friedel, M. A. Popp, J. C. J. Matern, Emerich M. Gazdag, I. V. Thiel, G. Volkmann, W. Blankenfeldt and H. D. Mootz, Chem. Sci., 2019, Advance Article , DOI: 10.1039/C8SC01074A

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