Structural basis for proteintrans-splicing by a bacterial intein-like domain - protein ligation without nucleophilic side chains

A. Sesilja Aranko, Jesper S. Oeemig, Hideo Iwaï
2013 The FEBS Journal  
Protein splicing in trans by split inteins has become a useful tool for protein engineering in vivo and in vitro. Inteins require Cys, Ser or Thr at the first residue of the C-terminal flanking sequence because a thiol or hydroxyl group in the side chains is a nucleophile indispensable for the transesterification step during protein splicing. Newly-identified distinct sequences with homology to the hedgehog/intein superfamily, called bacterial intein-like (BIL) domains, often do not have Cys,
more » ... r, or Thr as the obligatory nucleophilic residue found in inteins. We demonstrated that BIL domains from Clostridium thermocellum (Cth) are proficient at protein splicing without any sequence changes. We determined the first solution NMR structure of a BIL domain, CthBIL4, to guide engineering of split BIL domains for protein ligation. The newly-engineered split BIL domain could catalyze protein ligation by trans-splicing. Protein ligation without any nucleophilic residues of Cys, Ser and Thr could alleviate junction sequence requirements for protein trans-splicing imposed by split inteins and could broaden applications of protein ligation by protein trans-splicing. Database The resonance assignments and structure coordinates have been deposited in BMRB (18653) and RCSB (2LWY) Abbreviations Cth, Clostridium thermocellum; BIL, bacterial intein-like; GB1, the B1 domain of IgG binding protein G; Hh, hedgehog protein; Hint, hedgehog/intein; IMAC, ion-metal immobilized chromatography; IPTG, isopropyl thio-b-D-galactoside; Npu, Nostoc punctiforme. PDB, Protein Data Bank; PTS, protein trans-splicing; Ssp, Synechocystis sp. strain PCC6803; YFP, yellow fluorescent protein. 3256 FEBS Journal 280 (2013) 3256-3269 ª 2013 FEBS A. Sesilja Aranko et al. Cys/Ser/Thr-free protein ligation by BIL 3266 FEBS Journal 280 (2013) 3256-3269 ª 2013 FEBS Cys/Ser/Thr-free protein ligation by BIL A. Sesilja Aranko et al.
doi:10.1111/febs.12307 pmid:23621571 fatcat:at4hx2dyafha5hnnjytkjyv35u