Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing

Abstract

Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS.

Figure 1.

(A) Mechanism of class 1 intein splicing, with steps described in the text. Pho RadA variants used in this study and products from splicing, cleavage, and disulfide bonding reactions. Exteins, intein, and specific amino acid features of each protein are listed in the key.

Figure 2.

Pho RadA splicing stimulated by ssDNA: (A) splicing of WT RadA, (B) N-terminal cleavage of RadA N172A/T+1A, and (C) C-terminal cleavage of RadA C1A with or without ssDNA. For each panel, a representative gel is on the left and a graph of reaction kinetics on the right. For each graph, data are based on three independent experiments, with the error being the standard deviation. Reaction mixtures were incubated at 63 °C for the indicated times, separated by nonreducing SDS–PAGE, and stained with Coomassie. Levels of precursor (P), ligated extein (LE), intein–C-extein (I–C), N-extein–intein (N–I), and C-extein (C) proteins were measured by densitometry. Tris-EDTA (TE) was used as a buffer control for samples without ssDNA.

Figure 3.

ssDNA promotes disulfide between catalytic nucleophiles. (A) Gel demonstrating the internal disulfide bond between C1 and C +1 and influence of ssDNA. Pho RadA C+1 was purified under nonreducing conditions (–TCEP). Reaction mixtures were incubated at the indicated temperatures for 5 min, separated by SDS–PAGE, and stained with Coomassie. Levels of the oxidized precursor (P_OX) and reduced precursor (P_RED) were measured by densitometry. Tris-EDTA (TE) was used as a buffer control in the absence of ssDNA. (B) Quantification of C1–C+1 disulfide bond formation with or without ssDNA and TCEP prior to incubation (T₀) or after 5 min at 63 °C. Error bars represent the standard deviation from three independent experiments.

Figure 4.

Model of ssDNA-based acceleration of Pho RadA protein splicing. We propose that inhibitory interactions (dashed lines) between intein residue N172 and intein (D153) and extein (R+116 and R+119) residues and the distance between nucleophiles (bidirectional arrows) block splicing. Upon binding to ssDNA, inhibitory interactions are broken, allowing Asn cyclization and splicing to proceed (Figure 2). Additionally, we propose that ssDNA binding compresses the intein active site, bringing C1 and T+1 into the proximity for catalysis (Figure 3). The color scheme is the same as that in Figure 1.