Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein.
Fig: (a) The process of protein splicing and splicing side-reactions and (b) Comparison of intein sequences. (a) Protein splicing is facilitated by an intein, an intervening protein that interrupts the N-extein and C-extein. Splicing results in the ligation of the N-extein and C-extein and excision of the intein. Thiolysis or hydrolysis of the thioester formed in either steps 1 or 2 of splicing can result in N-terminal cleavage if the third step of splicing is blocked by mutation. Premature cyclization of the C-terminal Gln or Asn of the intein (step 3 of splicing), uncoupled from steps 1 or 2, can result in C-terminal cleavage. (b) A sequence comparison of the DNA PolII inteins from Pyrococcus abyssi (Pab) andPyrococcus horikoshii (Pho). The EMBOSS water tool was used to align the inteins. Identical residues are marked with a pipe and similar residues are marked with a colon. Intein blocks A and B make up the N-terminal segment of the active site, and blocks F and G make up the C-terminal segment of the active site. Blocks C, D, and E are missing as both inteins are so-called “mini-inteins” that have an NCR in place of a homing endonuclease domain.
We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.
Williams, J.E., Jaramillo, M.V., Li, Z. et al. An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein. Sci Rep 11, 11680 (2021). https://doi.org/10.1038/s41598-021-91090-w