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

Abstract

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.

Fig. 1. Mechanisms of protein splicing and the N–S acyl shift. (A) Mechanism of the protein splicing pathway of class 1 inteins. Shown here are Cys and Ser residues at the 1 and +1 positions, respectively, as present in the Ssp DnaB intein. Step 1: N–S acyl shift to the linear thioester intermediate; step 2: trans-esterification to the branched intermediate; step 3: asparagine cyclization to effect succinimide formation and cleavage of the C-terminal scissile bond; step 4: spontaneous O–N shift to the final splice product (uncatalyzed). (B) Side-reaction of N-terminal cleavage through nucleophilic attack of the linear thioester. This step is enforced when the C-terminal splice junction is mutated. (C) Mechanistic contribution of the essential block B histidine in the N–S acyl shift. (D) New artificial mechanisms to compensate for a lacking block B histidine as proposed in this work. Red numbers indicate the nomenclature in residue numbering at the extein–intein junctions.

Fig. 2. Extein dependence at the (–1) position of semisynthetic protein trans-splicing. (A) Scheme of the reactions involving the WT Ssp DnaB and M86 inteins. (B) Analysis of reactions on SDS-PAGE gels using Coomassie-staining (top) or UV illumination (bottom panel). Int^C proteins (20 μM) were incubated with the indicated peptides (60 μM) for 24 h and then quenched by SDS loading buffer and boiling. The variation of a single residue had a surprisingly large impact on the migration behavior of some of the splice products (SP). The weak bands visible for pep3 in the lower panels seem to result from a slight impurity of this peptide and were also observed in the absence of a protein partner (data not shown). (C) Yields of protein trans-splicing and C-terminal cleavage reactions determined by densitometric analysis of the Coomassie-stained gels shown in B. Error bars indicate standard deviations. Asterisks denote a protein contamination.

Fig. 3. Complex formation and N-terminal cleavage of split intein pairs blocked in the essential residues of the block B and G motifs. (A) Scheme of the reactions involving the triple mutants (H73A, N154A, S+1A) of the WT Ssp DnaB and M86 inteins. (B) Analysis of reactions on SDS-PAGE gels using Coomassie-staining (top) or UV illumination (bottom panel). Int^C proteins (20 μM) were incubated with the indicated peptides (60 μM) for 24 h and then quenched by SDS loading buffer without boiling. Asterisks denote a protein contamination.

Fig. 4. HPLC analysis of N-terminal cleavage with Int^C constructs containing triple mutations in the blocks B and G. Analyses of the Ex^N–Int^N peptides (Fl-KKESX-Int^N) are shown before (left panel) and after (middle and right panels) incubation with Int^C constructs. Absorption was measured at 280 nm, which detects only the Ex^N portion of the peptides (Fl-KKESX), but not the cleaved Int^N portion. Int^C proteins (15 μM) and peptides (30 μM) were incubated for 24 h. Samples were acidified with 0.1% TFA and boiled for Int^C precipitation prior to HPLC analysis. Note that the double peaks originate from the 5,6-isomers of the carboxyfluoresceine moiety. The identities of the cleaved Ex^N fragments were verified by MS-analysis (data not shown). The percentages of N-terminal cleavage are given in Table 1. The red boxes highlight the N-terminal cleavage products.

Fig. 5. Extein dependent compensation of the block B histidine in semisynthetic protein trans-splicing. Reaction schemes are as shown in Fig. 1A but using the H73A mutants of both inteins. (A) Analysis of reactions on SDS-PAGE gels using Coomassie-staining (top) or UV illumination (bottom panel). Int^C proteins (20 μM) were incubated with the indicated peptides (60 μM) for 24 h and then quenched by SDS loading buffer and boiling. The weak bands visible for pep3 in the lower panels seem to result from a slight impurity of this peptide and were also observed in the absence of a protein partner (data not shown). (B) Yields of protein trans-splicing and C-terminal cleavage reactions determined by densitometric analysis of the Coomassie-stained gels shown in A, except for values for pep1–3 and pep8, which were estimated from the fluorescence signals in the bottom panels. Error-bars indicate standard deviations. Asterisks denote a protein contamination.

Fig. 6. Extein dependence at the (–1) position of cis-inteins. (A) Reaction schemes. (B) Analysis of reactions on SDS-PAGE gels using Coomassie-staining (left panels) or Western-blotting (right panels). Shown are whole-cell lysates of E. coli cells expressing the cis-intein constructs for 3 h at 37 °C after induction with IPTG. Aliquots were mixed with SDS loading buffer and boiled.

Fig. 7. Extein dependent compensation of block B histidine of cis-inteins. Reaction schemes are as shown in Fig. 5A but using the H73A/N154A/S+1A mutant for the isolated N–S acyl shift resulting in N-terminal cleavage (panel A) and H73A mutants to allow for protein splicing (panel B), respectively, when the essential histidine residue is compensated for. Reactions were analyzed on SDS-PAGE gels using Coomassie-staining (left panels) or western-blotting (right panels) using whole-cell lysates of E. coli cells expressing the cis-intein constructs for 3 h at 37 °C after induction with IPTG. Aliquots were mixed with SDS loading buffer and boiled.

Fig. 8. Overlay of peptide backbones of intein crystal structures. (A) Overlay of the WT Ssp DnaB and the M86 intein precursors. (B) Overlay of the M86 intein with its extein(–1) and active site mutants. The endonuclease loop regions between amino acids 98 and 116 (indicated with arrows) and extein residues upstream of aa(-2) and downstream of aa(+2) have been omitted for comparability. These regions are illustrated in Fig. S2.

Fig. 9. Effect of mutations on the structure of the N-terminal splice junction. Shown are close-ups of two extein residues upstream and two intein residues downstream of the scissile peptide bond. Panel C additionally shows a stick representation of the block G histidine (H153).