Characterizing the Extein Dependence and Catalytic Mechanism of the Ultra-fast Split Intein NrdJ-1

Abstract

Split inteins are valuable biochemical tools due to their ability to undergo an autocatalytic process called post-translational splicing in which the intein fragments associate and selfexcise, ligating together the flanking N- and C-terminal residues, known as exteins, with a new peptide bond. While inteins are currently used in a variety of research applications, many naturally split inteins suffer from extein dependence and slow splicing kinetics. First characterized in 2009 and later applied to multiple studies in chemical biology, the ribonucleotide reductase class II (NrdJ) catalytic subunit NrdJ-1 is a promising ultra-fast split intein. NrdJ-1’s extein dependence and catalytic mechanism, however, have yet to be studied to determine the intein’s promiscuity and to discover integral non-catalytic residues that assist in protein splicing, called accelerators. Using established kanamycin and in vitro splicing assay methods from the Muir Lab, along with a newly generated Xray crystallography structure, the extein dependence of the -1 extein position and existence of accelerator residues within NrdJ-1 was explored. NrdJ-1 was found to have great promiscuity at the -1 extein position, exhibiting tolerance to most amino acids except for aspartate, proline, and valine. The presence of three accelerator residues: Y83 and Y89 (on IntN), and H39 (on IntC), were also discovered. They use pi-pi interactions and hydrogen bonds to facilitate association between intein fragments and promote fast splicing kinetics. The results demonstrate that NrdJ-1 is a promiscuous intein that can be fused with a variety of exteins at the IntN fragment for different research purposes. The presence of accelerator residues also enhances the knowledge of noncatalytic influences on intein splicing rates, allowing for the possibility of engineering faster versions of inteins that have historically suffered from slow splicing.