A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine.

Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (-1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(-1) residue, including d-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N-S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (-1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(-1)Phe and Gly(-1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein-intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein-intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.

Altered Coordination of Individual Catalytic Steps in Different and Evolved Inteins Reveals Kinetic Plasticity of the Protein Splicing Pathway.

Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N- S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step. The structure-activity relationship at the N-terminal scissile bond was studied with atomic precision using a semisynthetic split intein. We show that the removal of the extein acyl group from the α-amino moiety of the intein's first residue is strictly required and sufficient for the up-regulation switch. Even an acetyl group as the smallest possible extein moiety completely blocked the switch. Furthermore, we investigated the M86 intein, a mutant with faster splicing kinetics previously obtained by laboratory evolution of the Ssp DnaB intein, and the individual impact of its eight mutations. The succinimide formation was decoupled from the first step in the M86 intein, but the acquired H143R mutation acts as a brake to prevent premature C-terminal cleavage and thereby maximizes splicing yields. Together, these results revealed a high degree of plasticity in the kinetic coordination of the splicing pathway. Furthermore, our study led to the rational design of improved M86 mutants with the highest yielding trans-splicing and fastest trans-cleavage activities.

Development of a screening system for inteins active in protein splicing based on intein insertion into the LacZα-peptide.

Protein splicing by inteins has found diverse applications in biotechnology, protein chemistry and chemical biology. Inteins display a wide range of efficiencies and rates unpredictable from their amino acid sequences. Here, we identified positions T22S and S35 in the LacZα peptide as intein insertion sites that strictly require protein splicing, in contrast to cleavage side-reactions, to allow for complementation of ß-galactosidase activity. Both the cis-variant of the M86 mutant of the Ssp DnaB intein and a split form undergoing protein trans-splicing gave rise to formation of blue colonies in the ß-galactosidase read-out. Furthermore, we report the two novel, naturally split VidaL T4Lh-1 and VidaL UvsX-2 inteins whose N-terminal fragments consist of only 15 and 16 amino acids, respectively. Initial biochemical characterization with the LacZα host system of these inteins further underlines its utility. Finally, we used the LacZα host system to rapidly identify amino acid substitutions from a small randomized library at the structurally conserved intein position 2 next to the catalytic center, that are tolerated for protein splicing activity of the M86 intein. These findings demonstrate the potential of the system for initial testing and directed evolution of inteins.

Mapping of the Communication-Mediating Interface in Nonribosomal Peptide Synthetases Using a Genetically Encoded Photocrosslinker Supports an Upside-Down Helix-Hand Motif.

Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation domains is only poorly understood. Conflicting helix-helix and helix-hand models were previously proposed. Here, the genetically encoded photocrosslinker p-benzoylphenylalanine (BpF) was incorporated into the C-terminal communication-mediating domain (COM) of GrsA. Using the partner elongation module TycB1 to form a dipeptide product, we could correlate the ability to form covalent crosslinks with the functional module interaction. Perturbation of the module interaction with the large side chain of BpF in a scan at 19 positions demonstrated the importance of three hydrophobic residues in an α-helical arrangement. Mapping of covalent crosslinks using tandem mass spectrometry revealed the residues from the interior of the condensation domain as part of the protein interface; a finding not predicted by the helix-helix model. The epimerization domain of GrsA was found to be important for the interaction. Together with multiple sequence analyses and structural modeling, our results suggest an upside-down helix-hand model in which the C-terminal COM-helix is embedded in a hand motif with a hydrophobic core in a reversed orientation compared to a previous proposal. Our results provide a more detailed and the first direct structural understanding of the COM domain interaction and will contribute to successful biocombinatorial engineering attempts in the design of artificial NRPS templates.

Generation of a genetically encoded, photoactivatable intein for the controlled production of cyclic peptides.

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split-intein-mediated SICLOPPS technology provides a generic access to fully genetically encoded head-to-tail cyclized peptides and large libraries thereof (SICLOPPS=split-intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light-dependent intein using the photocaged tyrosine derivative ortho-nitrobenzyltyrosine incorporated at an internal, non-catalytic position is now reported. Stable intein precursors were purified from the E.coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.

Ligation of synthetic peptides to proteins using semisynthetic protein trans-splicing.

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

Chemical-tag labeling of proteins using fully recombinant split inteins.

Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.