Crystal structure of a thermophilic O6-alkylguanine-DNA alkyltransferase-derived self-labeling protein-tag in covalent complex with a fluorescent probe

https://doi.org/10.1016/j.bbrc.2018.04.139Get rights and content

Highlights

  • The labeling of proteins is a valid approach for studying their function.

  • SNAP-tag® is a self-labeling protein tag that binds different probes (e.g. SVG).

  • SsOGT-H5 is a thermostable alternative to the SNAP-tag® technology.

  • The SsOGT-H5-SVG structure reveals new details of protein-probe interaction.

  • The SsOGT-H5-SVG structure highlights new OGTs structure-function relationships.

Abstract

The self-labeling protein tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-tag® technology under harsh reaction conditions.

Here we present the crystal structure of SsOGT-H5 in complex with the fluorescent probe SNAP-Vista Green® (SsOGT-H5-SVG) that reveals the conformation adopted by the protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the protein active site. We gained new insights in the conformational changes possibly occurring to the OGT proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H5-SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H5-SVG structure, highlighting the role of the fluorescent ligand in keeping the protein stable after the trans-alkylation reaction.

Introduction

The fluorescent labeling of proteins is a powerful approach to study any aspects of the biology of the cell, including protein function, localization, trafficking and macromolecular interaction networks, both in vitro and in vivo. In many cases, the labeling is achieved by expressing a chimera, in which the protein of interest is fused to a polypeptide (i.e. tag) that behaves as the traceable moiety for protein imaging [1,2]. The self-labeling protein tags specifically and covalently bind different small-molecule fluorophores, a property that finds a huge number of applications [[3], [4], [5], [6], [7]]. A well-characterized example is the SNAP-tag® (New England Biolabs) [8], an engineered form of the hAGT, responsible for alkylated-DNA direct repair [9,10]. AGTs (EC: 2.1.1.63; alternative names: MGMT or OGT) are small suicidal proteins that perform the stoichiometric transfer of the alkyl group from the O6 position of an alkylated-guanine (or from the O4 position of an alkylated-thymine) to the catalytic cysteine residue of the protein active site [11]. The single-step trans-alkylation reaction brings the base back to the original state, while leaving the protein permanently inactivated and more prone to destabilization and degradation [12,13]. The SNAP-tag® was obtained through a directed protein evolution approach, by introducing 19 amino acid substitutions in the hAGT sequence and by deleting the 25 residues at the C-terminus of the protein [9,10,14]. This engineered protein reacts with BG derivatives bearing a fluorescent substituting group at their O6 position, such as the SVG reagent [9]. Compared to the parental hAGT the SNAP-tag® displays an enhanced activity and a remarkably improved stability both in vitro and when expressed inside the cell. Conversely, the SNAP-tag® does not possess any significant DNA-binding or alkylated-DNA repairing activity, thus satisfying a mandatory requirement to avoid an improper localization or an altered function of the SNAP-tag-fused protein of interest [10].

The SNAP-tag® is generally used for studying mesophilic species [10], whereas a thermostable OGT-based protein-tag could offer a greater exploitability to characterize organisms which are exposed to harsh growth conditions, and a superior intrinsic robustness in in vitro experiments requiring a wide range of temperature, pH, ionic strength and the presence of common denaturing agents [15,16]. To this end, an engineered variant of the Sulfolobus solfataricus OGT was previously prepared (hereon indicated as SsOGT-H5), which is characterized by the presence of a number of amino acid substitutions in the catalytic C-terminal domain of the protein. In detail, 5 mutated residues map at the DNA HTH motif (i.e. S100A, R102A, G105K, M106T and K110E), and on residue maps at the C-side of the active site loop (S132E), thus only partially matching the point-mutation profile of the hAGT-derived SNAP-tag® [10,15,16]. Nonetheless, thus mutagenesis approach completely abolished the SsOGT-H5 variant capability to bind the DNA, while preserved the protein self-labeling efficiency in the presence of the SVG probe [15,16]. The SsOGT-H5 properly works as a self-labeling protein-tag both in the mesophilic organism E. coli, as well as in thermophilic species as the bacterium Thermus thermophilus and the archaeaon Sulfolobus islandicus [16,17].

Although the SNAP-tag® and the SsOGT-H5 proteins have been extensively characterized, a structural snapshot of the protein architecture upon the trans-alkylation reaction with a synthetic probe was missing. We solved and report here the crystal structure of the SsOGT-H5 protein in covalent complex with the O6-substituting group of the SVG reagent (abbreviated as SsOGT-H5-SVG). Beside representing the first crystal structure of a AGT-derived self-labeling protein-tag in complex with a fluorescent probe, our data provide a structural overview of the amino acids that participate in the coordination of the bulky adduct in the post-reaction state of the protein, including an unprecedented role of residues belonging to the poorly functionally characterized N-terminal domain. Moreover, by performing molecular dynamics simulations on the novel SsOGT-H5-SVG structure, we gained insights into how the overall stability of the SsOGT-H5 protein could be affected by the presence of the fluorescent moiety in the protein active site. Overall, our analysis could be used for further improving the selectivity of the corresponding SsOGT-H5-tag/fluorescent substrate system.

Section snippets

Chemicals

All reagents were obtained from Sigma-Aldrich unless otherwise specified.

Protein expression and purification

The SsOGT-H5 variant was expressed in the E. coli ABLE-C strain and purified as previously described [15,16].

Crystallization and data collection

In order to prepare the self-labeled SsOGT-H5 protein to be used in crystallization experiments, a solution of the commercial SVG reagent (250 μM) was mixed with the SsOGT-H5 protein solution (10 mg/mL in 20 mM phosphate buffer, pH 7.3 and 150 mM NaCl) at a protein:SVG molar ratio of 1:1, and incubated overnight at

Structural analysis of the SVG-labeled SsOGT-H5 variant

SVG-labeled SsOGT-H5 produced crystals of remarkable quality that diffracted at 2.0 Å resolution, indicative of the structural integrity of the ligand-protein covalent adduct. Notably, the high-quality electron density map allowed us to model the SVG-Cys119 covalent adduct resulting from the trans-alkylation reaction, and to unambiguously assign the conformation adopted by the carboxyfluorescein diacetate N-succinimidyl ester moiety of the ligand to the para-isomer (Fig. 1, inset). As described

Funding

This work was supported by Fondazione Cariplo (Ricerca biomedica condotta da giovani ricercatori, project 2016-0604).

Acknowledgments

The authors acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation at beam line ID23-2. We gratefully acknowledge the support of NVIDIA Corporation with the donation of the Titan X Pascal GPU used for this research.

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