First thermostable CLIP- tag by rational design applied to an archaeal O6 -alkyl-guanine-DNA-alkyl-transferase

Abstract

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O⁶ -benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC⁸ . The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC⁸ was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O⁶ -alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant.

Keywords: AGT, OGT, MGMT, O6-alkyl-guanine-DNA-alkyl-transferase; BC, O2-benzyl-cytosine; BG, O6-benzyl-guanine; HTH, helix-turn-helix motif; IMAC, immobilized metal affinity chromatography; Orthogonal substrate specificity; Protein engineering; Protein labelling; Protein-tag; SLP, Self-Labelling Protein-tag; Thermozymes.

Graphical abstract

Fig. 1

Scheme of the irreversible reaction of SNAP- and CLIP-tag on their respective BG- and BC-derivative substrates. Upon the reaction, the benzyl moiety is covalently linked to the catalytic cysteine (white triangle). If desired chemical groups (indicated as 1 and 2) are previously conjugated to BG- and BC-, it is possible to achieve a specific multi-protein labelling by these commercial SLPs.

Fig. 2

Commercial and thermostable SLPs. Cartoon representation of SLPs, with BG- and BC- substrate specificity coloured as indicated in the legend.

Fig. 3

Molecular contacts gained by SsOGT^CLIP with respect to Chimera^CLIP.Fig. 3a represents the ion pair between E78 and K81; Fig. 3b shows the N_ter-to-C_ter contact based on H-bond of W84 with I58; Fig. 3c highlights the hydrophobic interaction between L147 and F53. Each panel is indicated with the corresponding letter (in red) in the cartoon representing the optimal superposition of SsOGT^CLIP and Chimera^CLIP predicted structures, depicted in orange and grey, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Substrate specificity of CLIP-tag and SsOGT-MC⁸ by immobilization studies. a) A fixed amount of protein (indicated as input, I) is added to a specific amount of a BG-resin: if the covalent reaction occurs (green enzymes), the resin captures the protein reducing the amount of non-bound protein (indicated as flow-through, FT), as measurable after SDS-PAGE analysis. On the contrary, orthogonal activity (red enzymes) does not allow the covalent binding of the protein on the resin. The value of the specific covalent binding is calculated by the equation shown in the figure; b) SDS-PAGE and histogram of the relative efficiency of binding of commercial and thermostable SLPs. Gels are an example of three independent experiments for each protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

In vivo heterologous expression of ^TSSNAP and ^TSCLIP in the thermophilic bacterium T. thermophilus HB27^EC. The ogt-H⁵ and ogt-MC⁸ genes were cloned into the pMK184 shuttle vector for their heterologous expression in HB27^EC strain. Overnight cultured cells were washed and incubated at 65 °C for 1 h in the presence of 1.0 μM fluorescent substrates (as indicated) and then subjected to SDS-PAGE (further details are described in the Section 5.4). The HB27^EC/pMK184 strain was used as negative control, as well as 1 μg of each purified protein was used as positive control. In the H⁵ lane was also loaded the protein marker.

Fig. 6

Molecular contacts gained by ^TSCLIP with respect to SsOGT^CLIP.Fig. 6a is a zoom-in of the network of contacts coordinated by Y90 in which we highlighted the potential H-bond between Y90 and the main chain of P130, V122 and the side chain of K138 respectively. Fig. 6b represents the H3 intra-helix ion pair between D95 and K91 and the H-bond established by S96 and E78; the latter substitutes the intra-helix contact of SsOGT^CLIP (orange) with an inter-helix bond in ^TSCLIP (raspberry). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Surface representation of crystal structures of SsOGT and ^TSCLIP bound to the BC- and BG-derivatives.Fig. 7a refers to ^TSCLIP (raspberry) bound to BC (on the left) and BG (on the right) with a zoom on the active site entrance showing the difficult accessibility of BG imidazole moiety; Fig. 7b refers to the same comparison performed on wild type SsOGT (cyan) (representative of ^TSSNAP) highlighting the wider active site gate of this construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)