Recent progress in intein research: from mechanism to directed evolution and applications

Abstract

Inteins catalyze a post-translational modification known as protein splicing, where the intein removes itself from a precursor protein and concomitantly ligates the flanking protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and protein chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous protein contexts, as well as novel applications of inteins for cell biology and protein engineering, which were made possible by a clearer understanding of the protein splicing mechanism.

Fig. 1

Intein-mediated protein splicing. Schematic representation of the standard protein splicing mechanism, which proceeds in four steps: (1) N–X acyl shift, (2) trans-(thio)esterification, (3) asparagine cyclization, and (4) X–N acyl shift. The first residue of standard (class 1) inteins can either be Cys or Ser, whereas the first residue of the C-extein can be Cys or Ser, as well as Thr

Fig. 2

Intein types found in nature and their similarity to hedgehog. a The three different intein types are shown schematically in the context of an N- and C-terminal extein, where I^N and I^C represent the N- and C-terminal intein splicing domains, respectively. ENDO corresponds to the homing endonuclease domain found exclusively in maxi inteins. The C-terminal domain of the hedgehog protein (Hh-C) is homologous to the splicing domain of inteins; in this case, the N-terminal domain (Hh-N) and a cholesterol molecule function as “exteins”. b The processing mechanism of hedgehog is initiated by an N–S acyl shift between the first residue of Hh-C (Cys) and the preceding peptide bond, as is the case in many inteins (see Fig. 1). The thioester bond is then attacked by the hydroxyl group of a cholesterol molecule, which results in the esterification of cholesterol to the C-terminus of Hh–N

Fig. 3

Intein sequence motifs and splicing mechanisms. a Schematic illustration of a mini-intein with the relative locations of the conserved sequence motifs (A, B, F, and G) are shown on top. Nomenclature of amino acid numbering in inteins is indicated (e.g., −1 is the last residue of the N-extein, and +1 is the first residue of the C-extein). The consensus sequences of all motifs are shown for the three intein classes [12] with bold-faced, uppercase letters representing highly conserved residues, and regular uppercase letters indicating moderately conserved residues. A “-” symbol is given where acidic residues are conserved, “h” stands for hydrophobic residues, “n” for nucleophilic residues, and “.” if there is no significant conservation at a position. The asterisks above residues in class 3 highlight the conserved WCT triplet. The numbers below the consensus sequences indicate the nomenclature for numbering of residues within the motifs according to Tori et al. [12], which is also used throughout the text. b Splicing mechanisms of the three intein classes. Splicing of class 1 inteins begins with an N–S acyl shift (if first residue is a Cys) (step 1), followed by trans-esterification of the N-extein to the first residue of the C-extein (step 2). Cyclization of the last intein residue cleaves the intein off of the esterified exteins (step 3), and a spontaneous S–N acyl shift forms the peptide bond between the exteins (step 4). Class 2 inteins forego step 1 of class 1 inteins and directly attack the N-terminal scissile peptide bond with the first C-extein residue (step 1/2a). Class 3 inteins use an internal Cys residue to form a branched intermediate unique to class 3 inteins (step 1b), which is subsequently attacked by the first residue of the C-extein (step 2b), resulting in the canonical branched ester intermediate

Fig. 4

The initial N–X acyl shift in protein splicing of class 1 inteins. a Schematic representation of the peptide bond rearrangement to a (thio)ester bond at the N-terminal splice junction. The intein is represented by a thick line, whereas the N- and C-exteins are indicated by black circles. The scissile peptide bond with the A:1 residue (Cys in this case) as well as the G:7 and G:8 residues (Asn and Ser, respectively) are shown, along with possible mechanisms that allow the N–S acyl shift to occur. b The pK _a shift mechanism during the initial N–S acyl shift as proposed by NMR and quantum mechanical/molecular mechanics calculations, where the B:10 His performs acid–base catalysis with the help of a water molecule [40]. c Proposed deprotonation of the A:1 N-nucleophile (Cys) by the F:4 Asp residue during N–S acyl shift [46]

Fig. 5

The trans(thio)esterification step as catalyzed by class 1 inteins. a Schematic representation of the intein active site after the N–S acyl shift (left), where a base might potentially activate the G:8 C-nucleophile for attack of the thioester bond at the N-terminal splice junction. This attack yields a tetrahedral intermediate, which is likely stabilized by an oxyanion hole. Acid-catalysis then protonates the A:1 side chain, resulting in the branched ester intermediate. b Possible role of the F:4 Asp residue during the trans-thioesterification reaction by activating the G:8 C-nucleophile [54]

Fig. 6

The final step in protein splicing catalyzed by all intein classes, Asn cyclization. a Schematic representation of the active site, starting with the branched ester intermediate, where base-catalysis in combination with ground-state destabilization of the C-scissile peptide bond possibly assist the nucleophilic attack of the G:7 Asn side chain on the peptidyl carbon. The resulting tetrahedral intermediate is likely stabilized by an oxyanion hole, and acid-assisted protonation of the peptidic nitrogen leads to cleavage of the C-scissile peptide bond. b Proposed scheme for Asn cyclization in a low-pH environment as deduced by QM/MM calculations [62]. c Proposed scheme for Asn cyclization derived by a computational model as based on the Ssp DnaB mini-intein crystal structure [66]

Fig. 7

The final, uncatalyzed reaction in protein splicing (X–N acyl shift). The formation of the peptide bond between the exteins from the ester bond is not catalyzed by the intein but represents a spontaneous rearrangement to the more stable amide linkage

Fig. 8

Molecular evolution of inteins. a ClustalW alignment of amino acid sequences from inteins that have been evolved by error-prone PCR and selected for more efficient splicing under various selection pressures. The conserved intein motifs are indicated above the sequences, and secondary structural elements are highlighted (blue boxes β-strands, orange boxes α-helices). In the sequence of Mtu RecA-ΔΔI_hh, the black arrows indicate the position of the hedgehog (hh) protein-derived β-turn sequence (VRDVETG), which replaces residues 95–402 of the wild-type Mtu RecA intein including the endonuclease domain [78]. In the chimeric Npu/Ssp DnaE intein sequence, the black arrow indicates the boundary of the split intein fragments (residues 1–102: N-intein from Npu, residues 103–138: C-intein from Ssp). In the Ssp DnaB mini-intein sequence, the black arrow indicates the deletion point (∆) of the endonuclease domain [81]. Residues below the sequences indicate mutations introduced by molecular evolution. For the Mtu RecA-ΔΔI_hh intein, the following mutations were also found but omitted for simplicity: D24E, D24N, K74M, R96C, R96P. The F120 residue is often referred to as F421 in the literature (in accordance with the numbering of the wild-type maxi Mtu RecA intein). In the case of the Npu/Ssp DnaE intein, red residues correspond to mutations isolated during selection for a different splice junction context, whereas green residues indicate mutations discovered when the selection pressure was temperature (37°C). Leu15 was either mutated to Ile or Ser, depending on the experiment. b The locations of the mutations shown in a were mapped onto the structures of the parent inteins using PyMol. PDB codes: Mtu RecA-ΔΔ_hh, 2IN9 [39]; Npu DnaE, 2KEQ [108]; Ssp DnaB mini, 1MI8 [36]

Fig. 9

Split intein-based labeling of recombinant proteins. Recombinant proteins can be modified with chemical groups (represented by a star) either at the N- or C-terminus using semisynthetic protein trans-splicing

Fig. 10

Controlling intein function with photoremovable groups. a Installing two 6-nitroveratryl (Nvl) groups at positions Gly19 and Gly31 in the Ssp DnaE I^C split intein fragment disturbs the interaction with the cognate I^N fragment. The high affinity association of the intein fragments and subsequent protein trans-splicing is restored only upon irradiation with light (λ_365 nm). b Modification of the penultimate residue in the Ssp DnaE I^C fragment (here: Ala[G:7]Ser) with an o-nitroveratryloxycarbonyl (Nvoc) group still allows for split intein association but significantly perturbs the active site. Protein trans-splicing commences after treatment of the complex with light (λ_325nm), which results in deprotection and O–N acyl migration to properly rebuild the intein active site. c To engineer a semisynthetic version of the Ssp DnaB intein into a light-responsive switch for C-terminal cleavage, the 11-aa I^N fragment was rationally redesigned to contain (1) the cysteine isoster diamino propionic acid at position A:1 with the N-extein sequence X linked to the side chain, thereby resembling the linear thioester intermediate after the N–S acyl shift, and (2) an Nvoc-protected N-terminus. Liberating a free N-terminus by light-irradiation (λ_365nm) restores split intein association and C-cleavage activity