6FRH

Crystal structure of Ssp DnaB Mini-Intein variant M86

Summary for 6FRH

• Entry DOI: 10.2210/pdb6frh/pdb
• Related: 6FRE 6FRG
• Descriptor: Replicative DNA helicase,Replicative DNA helicase (2 entities in total)
• Functional Keywords: intein, protein splicing, hint domain fold, pre-splicing, splicing
• Biological source: Synechocystis sp.; More
• Total number of polymer chains: 2
• Total formula weight: 37773.52

Authors

Popp, M.A.,Blankenfeldt, W.,Gazdag, M.E.,Matern, J.J.C.,Mootz, H.D. (deposition date: 2018-02-15, release date: 2019-01-30, Last modification date: 2024-01-17)

Primary citation

Friedel, K.,Popp, M.A.,Matern, J.C.J.,Gazdag, E.M.,Thiel, I.V.,Volkmann, G.,Blankenfeldt, W.,Mootz, H.D.
A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine.
Chem Sci, 10:239-251, 2019

PubMed 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 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 -splicing split intein and the -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.

PubMed: 30713635
DOI: 10.1039/c8sc01074a

Experimental method

X-RAY DIFFRACTION (2.03 Å)