The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from acis-splicing intein

Abstract

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool.The 1.0 Å-resolution crystal structure of a variant from naturally split gp41-1 intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N-and C-terminal intein fragments. We successfully created orthogonal split inteins by engineering a similar charge network in the same region of a cis-splicing intein. The same strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins. KeywordsIntein, split intein, protein splicing, crystal structure, gp41-1, protein engineering

Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

“…This observation might indicate that the negative charge at the position corresponding to E71 in Pho RadA could be unfavorable for the splicing efficiency in many inteins. It is also noteworthy that not only the −1 and +2 positions but also the −2 and +3 positions could synergistically influence protein‐splicing efficiency, presumably by defining the local conformation due to the interactions between them [28,40]. We also observed the same effect from other residues than at the −1 and +2 positions on protein splicing by the gp41‐1 intein, pointing out that the −1 and +2 positions are not the only determinants for the splicing efficiency [28].…”

“…T51 of Npu DnaB Δ290 is presented in a stick model on the cartoon of the Npu DnaB Δ290 structure (PDB: 4O1R) [16], which corresponds to E71 in the structure of Pho RadA intein. Similarly, S42 of gp41‐1 intein structurally corresponds to E71 in the structure of Pho RadA intein, presented in a stick model (PDB: 6QAZ) [28]. These three residues are colored in green.…”

“…The comparison between Pho RadA and gp41‐1 indicated that E71 in Pho RadA could correspond to S42 in the structure of gp41‐1 (Fig. 7) [20,28]. The inspection of the substrate‐specificity profiles and three‐dimensional structures implies that it might be feasible to identify the common structure–function relationship among different inteins, even though the junction sequence dependency profile seems to be unique to individual inteins.…”

“…It is also noteworthy that not only the À1 and +2 positions but also the À2 and +3 positions could synergistically influence protein-splicing efficiency, presumably by defining the local conformation due to the interactions between them [28,40]. We also observed the same effect from other residues than at the À1 and +2 positions on protein splicing by the gp41-1 intein, pointing out that the À1 and +2 positions are not the only determinants for the splicing efficiency [28]. Not only the AA-type but also local structures in the presence of exteins and conformational dynamics such as flexibility could play essential roles in protein splicing [9,40].…”

“…The gene of the gp41‐1 intein was amplified from pBHDuet37 by PCR with two oligonucleotides I785 and I786 [28]. The gp41‐1 gene was digested with Bam HI and Kpn I and cloned into pLLSDuet1, resulting in the donor plasmid pLKDuet7.…”