Structural basis for protein trans‐splicing by a bacterial intein‐like domain – protein ligation without nucleophilic side chains

Abstract

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, Ser, 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. DatabaseThe resonance assignments and structure coordinates have been deposited in BMRB (18653) and RCSB (2LWY)

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

“…1A) has been seen in all intein structures solved by NMR and x-ray crystallography (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The fold comprises primarily ␤-sheets, loops, and two short helices and has pseudo two-fold symmetry (Fig.…”

“…Different inteins have evolved to splice different proteins in nature, and thus their extein sequence preferences are roughly defined by their native contexts. Deviation from these prefer- 2 The abbreviation used is: BI, branched intermediate. (19).…”

Intermolecular domain swapping induces intein-mediated protein alternative splicing

“…Additionally, inteins are flexible enzymes that can achieve protein splicing through diversified mechanisms 41 . Variations of protein-splicing mechanisms by intein and intein-related sequences are still increasing 41,42 . As more extensive genomic, metagenomic and proteomic data are available at an increasingly rapid pace, we anticipate more inteins and intein-related sequences as well as diversified natural and contrived molecules produced by iPAS, which could advance our understanding of their biochemical and physiological roles and their biotechnological applications.…”

Protein Splicing: How Inteins Escape from Precursor Proteins

“…BILs lack the C-extein +1 nucleophile and are therefore unable to form the block G branched intermediate (36–38). Both class 2 and class 3 inteins can still splice, although they lack a Ser 1 or Cys 1 nucleophile and are thus unable to form the linear (thio)ester intermediate (Fig.…”

“…Intein splicing domains may have been derived from ancient enzymes because they are small and are closely related by structure, conserved motifs, and enzymatic activities to Hedgehog autoprocessing domains, which activate essential signaling proteins for metazoan development (34, 35). Inteins are also related to b acterial i ntein- l ike (BIL) 2 domains (36–39). …”