Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications

Abstract

Here we describe self-splicing proteins, called inteins, that function as redox-responsive switches in bacteria. Redox regulation was achieved by engineering a disulfide bond between the intein's catalytic cysteine and a cysteine in the flanking 'extein' sequence. This interaction was validated by an X-ray structure, which includes a transient splice junction. A natural analog of the designed system was identified in Pyrococcus abyssi, suggesting an unprecedented form of adaptive, post-translational regulation.

Figure 1

Engineered redox-responsive intein precursor in vivo and in vitro. (a) Scheme for intein splicing. Conserved terminal intein residues are indicated as is the extein C+1 residue. N-extein and C-extein residues are designated by minus and plus signs, respectively. (b). FRET-based intein reporter with the DnaE intein (red) inserted between cyan and yellow fluorescent proteins. (c). Biomimetic Cys-3 variant, ^CPGCDnaE, displays enhanced redox sensitivity in vivo. Activities of ^CPGCDnaE and ^APGCDnaE inteins were quantified as the percentage of precursor (C-I_a-Y) remaining, 7 h and 18 h after induction of protein expression in E. coli DHB4, AD494 and origami. Data are representative of ≥2 independent experiments. Additional bands, observed previously with unboiled samples, were excluded from the quantitation (d). Effect of redox conditions on the in vitro cleavage kinetics of ^APGCDnaE and ^CPGCDnaE. N-extein cleavage was monitored by FRET loss that occurs upon cleavage by DTT or by hydroxylamine (HA). Each data point is an average of three measurements.

Figure 2

Crystal structure of the ^CPGCDnaE intein. (a). Ribbon diagram showing the intein (red) and the N-terminal extein (green). Magnified view of the Cys1-to-Cys-3 disulfide loop is shown in the box (b). Conformation of the CPGC disulfide loop. Comparison of the structure of the disulfide loop in the ^CPGCDnaE intein (red) with the two other observations of this loop in the Protein Data Bank (1JBQ, green; 1M6Y, cyan). Magnified view shows Gly-Cys peptide bonds, with an upward arrow pointing to the carbonyl oxygen atoms and a downward arrow pointing toward the amide nitrogen atoms. (c). Active-site interactions in the ^CPGCDnaE intein (intein, red; extein, green). Thr69 of the conserved TXXH motif contacts the amide nitrogen of Cys1 and the carbonyl oxygen of Pro-2 while His72, also of the TXXH motif, does not interact directly with the N-extein. Asp140 makes a water-mediated contact with the −1 carbonyl and a hydrogen bond with His147, also a conserved residue. Suc 159 is the C-terminal succinimide.

Figure 3

Protein splicing by the P. abssyi MoaA intein is redox sensitive (a). Schematic of ^CWYCMoaA precursor. Splicing of the MoaA intein (red) with native N-extein residues (circles) ligates the cyan fluorescent protein to a hexahistidine tag (His₆). (b) and (c). Suppressed activity of the ^CWYCMoaA intein in the absence of a reducing agent. Intein autoprocessing was monitored in the presence and absence of the reducing agent, TCEP, at 22 °C (squares) and 55 °C (triangles). Precursor (CFP^CWYCMoaA-His₆) and splicing product (CFP-His₆) were separated by nonreducing SDS-PAGE and detected by in-gel fluorescence (excitation 457 nm, emission 526 nm). Plot of precursor disappearance in panel c, using data of panel b, shows that TCEP is required for efficient ^CWYCMoaA processing at 22 °C and at 55 °C. Error bars represent s.d., n=3. (d). ^CWYCMoaA displays redox regulation in E. coli. Representative gel images showing CFP-fused MoaA precursor with wild-type Cys-3 (CFP^CWYCMoaA-His₆), and CFP-products remaining after 3 h of induction in DHB4 (left lane), AD494 (middle lane), and origami (right lane). (e). Cys-3 is required for MoaA precursor accumulation in origami. Graph was derived from images as in panel (a) and shows percent of unspliced CFP^CWYCMoaA-His₆ (C, light shaded) and CFP^AWYCMoaA-His₆ (A, dark shaded) precursors after expression in the indicated strains, averaged over three independent trials. Error bars represent s.d.