Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

Abstract

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of nonredundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides

“…For our project, we have chosen to utilize a protein-trans splicing (PTS) technique for bioconjugation. PTS is highly versatile and has numerous applications in protein engineering and chemical biology due to the unique pathway for the modification and cleavage of precursor proteins [31]. Protein splicing involves the modification of a protein precursor by removing the intein (intervening protein) and linking the exteins (external proteins) via a peptide bond to form the mature protein structure.…”

Gold Nanoparticles Augment N-Terminal Cleavage and Splicing Reactions in Mycobacterium tuberculosis SufB

“…Inteins are intervening polypeptides that auto-excise from host proteins, bonding the neighboring exteins in a process termed protein splicing ( Perler et al, 1994 ; Paulus, 2000 ; Wu et al, 2014 ; Lennon et al, 2016 ; Lennon et al, 2018 ; Hoffmann et al, 2021 ). Protein splicing commences with a series of nucleophilic displacement reactions—an N/S or N/O acyl shift, a transesterification reaction, Asn cyclization, and an S/N or O/N acyl shift ( Figure 1A ) ( Volkmann et al, 2013 ; Topilina et al, 2015 ; Nanda et al, 2020 ).…”