Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Abstract

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoelike pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of inteinbased technologies.Protein splicing is a posttranslational modification, a multistep autoprocessing event, where intervening proteins (inteins) self-excise themselves from the precursors followed by the ligation of external proteins (exteins) (1). Once thought anomalies, inteins have since been found in all domains of life. Beyond their biological context, inteins are particularly intriguing as their capacity to process the polypeptide backbone makes them useful tools for protein engineering. Despite the fact that all inteins share the same fold and have highly conserved sequence motifs in their active sites, inteins have surprisingly different splicing efficiencies, and their extein sequence preferences differ dramatically. Here, we review studies on intein structure and dynamics that shed light on their complex conserved fold and their divergent efficiency and substrate specificity. The Intein FoldThe conserved horseshoe-like fold of inteins (Fig. 1A) has been seen in all intein structures solved by NMR and x-ray crystallography (2-11). The fold comprises primarily ␤-sheets, loops, and two short helices and has pseudo two-fold symmetry (Fig. 1B). Given this symmetry, it has been proposed that this fold arose due to a gene duplication event of some parent protein (6). The intein fold has three remarkable features: 1) the topology is complex, involving multiple passes of the polypeptide chain back and forth between the symmetry-related halves (Fig. 1B); 2) the extein-bearing termini of the intein are brought in close proximity (Ͻ10 Å) for splicing; and 3) multiple protease-like active sites composed of conserved sequence motifs are built around these termini to carry out each of the chemical steps involved in protein splicing. Folding of an intein is coupled to the reaction; the initial structure facilitates the first step of splicing, and thereafter each splicing step causes local conformational changes, affecting the fold of the catalytic apparatus and hence affecting the reaction coordinate and shifting the equilibrium positi...

Protein Splicing: How Inteins Escape from Precursor Proteins

“…Only the Sce VMA intein (86) and the Pho RadA intein (46) structures have distances between the C-extein nucleophile and N-terminal scissile bond that are directly compatible with catalysis (3.8 Å). This distance is much larger (∼8 Å) in all other intein structures to date and requires a conformational change for catalysis (41). A conformational shift was also proposed in the class 2 Mja KlbA intein where a rearrangement of Ser G:6 , Asn G:7 , and Cys +1 (G:8) backbone torsional angles could enable a close approach of the Cys +1 nucleophile to the N-terminal scissile bond (76).…”

“…Instead, we will discuss both the canonical and the alternative protein splicing mechanisms, and general strategies for catalysis and coordination of the steps. Detailed mechanism reviews are available (8, 9, 40), and intein structure, molecular dynamics, evolution, and applications are covered in companion reviews in this series (21, 33, 41). …”

Dynamics differentiate between active and inactive inteins

“…The optimization of intein function needs a delicate balance of protein structure, stability and conformational dynamics, explaining why mutations observed in evolved inteins are difficult to rationalize [37]. It is important to understand the structural and dynamical aspects of inteins for intein engineering and the improvement of intein-based technologies[38]. …”

Evolution, Mechanisms, and Applications of Intein-mediated Protein Splicing

“…Inteins share a structural fold with the essential metazoan Hedgehog signaling protein autoprocessing domain, suggesting that inteins evolved from ancient proteins that predate the separation of prokaryotes and eukaryotes (2, 3). Many modern intein genes are mobile genetic elements encoding intein proteins with a homing endonuclease domain that is able cleave DNA at the site at which the intein coding sequence is inserted.…”

“…The third minireview in this series, by Eryilmaz et al (3), explores the mechanism of protein splicing from a structural and molecular dynamics perspective and reports that, in native systems studied to date, splicing appears to occur as soon as the intein folds. This minireview also discusses the theme of trans- splicing with split precursors and its implications for nucleation of folding in all inteins.…”