Rapid Report pubs.acs.org/biochemistry
Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum Julie N. Reitter, Christopher E. Cousin, Michael C. Nicastri, Mario V. Jaramillo, and Kenneth V. Mills* Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610, United States S Supporting Information *
ABSTRACT: An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled Nterminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.
Figure 1. Schematic representation of protein splicing. Unspliced precursor protein (MIH) can promote protein splicing, resulting in the spliced product, MH, and the excised intein, I. Alternatively, the intein can promote N-terminal cleavage, with cleavage of the N-extein (M) from the intein−C-extein fusion (IH), uncoupled from splicing. Boxes are not shown to scale.
Cys must be reduced to initiate splicing; such inteins can serve as biosensors for the cellular redox state.12−15 Splicing also can be conditional on environmental state by oxidative or nitrosative damage to catalytic Cys residues or by resolution of inhibitory intein−extein interactions.16,17 Inteins also have been engineered to splice conditionally, including by the addition of small molecules, light, or a change in pH or temperature.18−30 As has been recently reviewed,31−38 protein splicing, and particularly conditional protein splicing, has wide applications in biotechnology, including protein purification, protein cyclization, site-specific labeling, biosensors, and gene delivery. A new, relatively mild method for promoting conditional splicing could lead to new and improved intein-based biotechnology methods. In this study, we show that the intein that interrupts the DNA polymerase II, large DP2 subunit from the extreme halophile Halobacterium salinarum sp. NRC1 (the Hsa PolII intein) can be expressed in E. coli and isolated as an unspliced precursor. The precursor can be induced to splice in vitro by incubation in the presence of a reducing agent and high concentrations of salt. We previously reported the protein splicing of the Methanocelleus marisnigri PolII intein (Mma PolII), which is conditional on cellular redox state.15 The Hsa PolII intein shares 35% residue identity with the Mma PolII intein and 46% similarity,39 particularly in conserved intein sequence blocks, which have 57% identity and 68% similarity (Figure S2), including a C-terminal Gln in place of the highly conserved Asn. This suggests that the Hsa PolII intein also should be able
P
rotein splicing is the process by which an intervening protein, or intein, facilitates its own cleavage from flanking polypeptides, or exteins, concomitant to extein ligation.1,2 Protein splicing usually is studied by overexpression of intein fusion proteins in Escherichia coli, in which most inteins are active and facilitate splicing. The canonical mechanism of protein splicing involves four steps:1,2 (i) an amide to ester or thioester rearrangement of the peptide bond linking the N-terminal extein (N-extein) and intein, (ii) a transesterification by which the N-extein is transferred from the side chain of the first residue of the intein to the side chain of the first residue of the C-terminal extein (Cextein), (iii) cleavage of the peptide bond linking the intein to the C-extein coupled to cyclization of the intein’s C-terminal Asn, and (iv) conversion of the ester or thioester linking the exteins to an amide and possible hydrolysis of the C-terminal aminosuccinimide of the excised intein to asparagine or isoasparagine (Figure S1). Coordination of the splicing steps is required to produce the ligated product; premature hydrolysis of the ester or thioester formed in step i or ii can result in cleavage of the N-extein from the fusion protein, uncoupled from splicing (Figure 1). It has not yet been shown if protein splicing plays a physiological role for the host organism. It is likely that potential intein-mediated regulation of the activity of flanking proteins would be conditional on a physiological cue. Inteins that display such conditional activity have been discovered. Inteins from thermophiles have been shown to be conditional on high-temperature incubation,3−10 while other inteins have native pH sensitivity.11 Both engineered and native inteins can have redox traps, by which a disulfide bond involving a catalytic © XXXX American Chemical Society
Received: February 12, 2016 Revised: February 25, 2016
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DOI: 10.1021/acs.biochem.6b00128 Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
Rapid Report
to promote protein splicing. A major difference between the two inteins is the presence of a highly charged region upstream of conserved intein block F in the Hsa PolII intein. In the structure of the Pyrococcus abyssi (Pab) PolII intein, which shares 23% residue identity and 39% similarity with the Hsa PolII intein, this region is part of a disordered loop.40 It also is likely that the Hsa PolII intein should facilitate splicing because activity of the uninterrupted DNA PolII subunit likely is essential to the cell, and because inteins invade many different proteins throughout the Halobacteria genus.41 To test the activity of the intein, we designed a plasmid to express a fusion protein with an N-extein of E. coli maltose binding protein, a short linker, and the 10 C-terminal residues of the native N-extein. This is fused in-frame to the 195-residue Hsa PolII intein and the C-extein, which consists of the six Nterminal residues of the native C-extein, a short linker, and a poly-His tag. We refer to this fusion protein as MIHHsaWT (Figure S3). Given that the C-terminal residue is Gln rather than the highly conserved Asn, we created a mutant fusion protein MIHHsaQN, which has a mutation of the intein’s Gln195 to Asn. We also created plasmids to express fusion proteins with mutations to the first residue of the intein (Cys1Ala, or MIHHsaC1A) or to the residues at the C-terminal splice junction (Gln195Ala/Cys+1Ala, MIHHsaQACA). However, unlike with the Mma PolII intein, we did not observe in vivo splicing with the Hsa PolII intein when it was expressed in E. coli between the same foreign exteins and observed no splicing upon protein purification (Figures 2 and 3, lanes P). Likewise, a comparison study surveying the activity of 20 inteins in E. coli reported no splicing of the Hsa PolII intein.42
Figure 3. Splicing as a function of an increasing NaCl concentration. Analysis of protein splicing via SDS−PAGE. Splicing was initiated by 28 °C incubation for 16 h of 2.8 μM purified fusion protein in buffer A supplemented with 2 mM TCEP and 5 mM EDTA, with the final salt concentration indicated. Lane “P” contained untreated protein.
surface.47,48 The Hsa PolII intein has 40 Asp or Glu residues of 195 total residues, with a pI of ∼4.4, as estimated by the ExPASy pI tool. Many H. salinarum proteins are destabilized and not active at low salt concentrations,44,49 whereas some are active at low salt concentrations50 or can have their salt dependence tuned by conservative surface residue changes or the introduction of disulfide bonds.51,52 Biophysical studies of the H. salinarum cysteinyl-tRNA synthetase document the influence of group 1 cations on the enzyme’s structure and thermal stability.53 Therefore, we attempted to induce splicing in vitro by high-salt incubation. The activity of the intein is dependent on salt concentration. Upon overexpression in E. coli and purification via affinity chromatography, we can isolate an unspliced precursor (Figures 2 and 3, lanes P). Upon incubation for 16 h at 28 °C, which is near the native growth temperature, MIHHsaWT promotes both splicing (conversion of MIH to MH and I, 22% in Figure 2) and N-terminal cleavage (conversion of MIH to M and IH, 24% in Figure 2) as a function of salt concentration (Figures 2 and 3). Incubation at higher temperatures promotes N-terminal cleavage to a greater extent than splicing (Figure S4). Proteins that include the intein migrate on SDS−PAGE in a manner inconsistent with their predicted molecular mass. This is not surprising, as proteins rich in negatively charged residues or with negatively charged or disordered motifs can display substantively aberrant migration by SDS−PAGE.54,55 To verify the identity of the SDS−PAGE bands, we used InVision HisTag in-gel stain, which specifically stains His-tagged proteins. The bands assigned to MIH, MH, and IH are stained, whereas those associated with M and I are not (Figure S5). Also, the reaction products were verified by MALDI-TOF MS, which confirms the m/z of the predicted bands with
