A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine
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Authors
Friedel, KristinaPopp, Monika A.
Matern, Julian C. J.
Gazdag, Emerich M.
Thiel, Ilka V.
Volkmann, Gerrit
Blankenfeldt, Wulf
Mootz, Henning D.
Issue Date
2018-10-03
Metadata
Show full item recordAbstract
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.Affiliation
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.Publisher
Royal Society of ChemistryJournal
Chemical ScienceAdditional Links
http://xlink.rsc.org/?DOI=C8SC01074AType
ArticleISSN
2041-65202041-6539
ae974a485f413a2113503eed53cd6c53
10.1039/C8SC01074A
Scopus Count
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