Characterisation of the ligand binding sites in the translocator protein TSPO using the chimeric bacterial-mammalian constructs

The translocator protein TSPO is in an important diagnostic and therapeutic target in a range of pathologies, including neuroinflammation and cancer. Despite the availability of several structures of TSPO homologues, our understanding of the molecular determinants that govern high-affinity interactions of TSPO with its ligands is incomplete. Here, in order to decipher the key structural elements of TSPO responsible for interactions with its ligands, we designed a panel of chimeric proteins mimicking the mammalian substrate binding site grafted onto the backbone of the Rhodobacter sphaeroides TSPO homologue, RsTSPO. One of the designed chimeric constructs, RsMouse, could be heterologously expressed and displayed improved binding affinities for the known TSPO drugs diazepam, PK11195 and NBD-FGIN-1-27. Furthermore, the chimeric protein had improved interactions with NBD-cholesterol, a fluorescent analogue of the presumed natural substrate of TSPO. Partial modifications of the transmembrane helix bundle in the chimeric construct differentially affected binding of the TSPO drugs and the natural substrates of TSPO, consistent with the presence of multiple ligand binding sites in the protein. Based on the available structures of TSPO homologues, the substrate interactions may involve a lateral opening of the protein in the TM1-3, and stabilisation of TM4-5 is important for drug-like ligand binding. These observations are consistent with our experimental results, which show that the determinants of high-affinity ligand interactions of TSPO are distinct for different classes of ligands.ISSN:1046-5928ISSN:1096-027

Expression and purification of the mammalian translocator protein for structural studies

<div><p>The translocator protein (TSPO) is an 18 kDa polytopic membrane protein of the outer mitochondrial membrane, abundantly present in the steroid-synthesising cells. TSPO has been linked to a number of disorders, and it is recognised as a promising drug target with a range of potential medical applications. Structural and biochemical characterisation of a mammalian TSPO requires expression and purification of the protein of high quality in sufficiently large quantities. Here we describe detailed procedures for heterologous expression and purification of mammalian TSPO in HEK293 cells. We demonstrate that the established procedures can be used for untagged TSPO as well as for C-terminally fused TSPO constructs. Our protocol can be routinely used to generate high-quality TSPO preparations for biochemical and structural studies.</p></div

Stable HEK293 cell line expressing a cleavable TSPO.

<p>Comparison of the best clones of bovine and pig TSPO using FSEC. Normalised to total protein concentration with Bradford assay, determined using the cell lysates. Pig TSPO shows a 2-fold higher expression compared to bovine TSPO and was therefore chosen for subsequent large-scale expression.</p

Overview of the screening procedures performed for homologue selection.

<p>(A) Small-scale expression test: in-gel fluorescence showing the expression levels of C-terminal GFP-10xHis constructs expressed in HEK293T cells and solubilised in 1% DM (1 human, 2 mouse, 3 rat, 4 dog, 5 bovine, 6 pig, 7 sheep, 8 chicken, 9 <i>Xenopus laevis</i>, 10 <i>danio rerio</i>, 11 <i>danaus plexippus</i>, 12 <i>drosophila melanogaster</i>). (B) Radioligand binding assay with [³H]PK11195 performed with all 12 C-terminally GFP-tagged TSPO homologues. (C) Thermostability of nine eukaryotic TSPO homologues assessed by incubating the in DDM solubilised proteins for one hour at a range of temperatures and subsequent analysis by FSEC. Control sample was incubated at 4°C (D) Small-micelle detergent screen of dog, bovine, pig and sheep TSPO. DDM-solubilised TSPO was diluted 10-fold into a buffer containing a secondary detergent, analysed by FSEC and the peak height was compared to the DDM-solubilised control sample.</p

Optimisation of the TSPO-YFP fusion constructs.

<p>(A) Construct design for TSPO-YFP fusions. The linker between the proteins was shortened with two amino acid steps. (B): Sequence alignment of linker region for all five fusion constructs. (C) Expression level of the best clone of the TSPO-YFP fusion constructs after stable cell line generation analysed by FSEC.</p

Large-scale purification of pig TSPO-YFP fusions.

<p>(A) Overview of the purification steps. (B) Coomassie blue stained SDS-PAGE gel and in gel fluorescence of protein at various stages of purification. The lanes are labelled as follows: “M”—molecular weight marker, “SN”—supernatant, “FT”—flow-through, “W1”—wash 1, “W2”—wash 2, “elu”–eluted fractions after affinity chromatography, “SEC”—pooled fractions after SEC. (C) Large-scale purification of TSPO-YFP fusion linker10aa in different detergents. Size-exclusion chromatography of pig TSPO fusion solubilised in DDM/CHS and subsequently purified in DDM/CHS (<i>left</i>), LDAO/CHS (<i>middle</i>) and DM/CHS (<i>right</i>). Peaks normalised to 25 g cells. (D) Final construct after purification. (E) Tryptophan quenching assay of TSPO-YFP with diazepam and PK11195 (n = 3). (F) Thermostability of the purified TSPO was assessed as described in “Materials and Methods”. Especially PK11195 has a stabilising effect on TSPO.</p

Sequence alignment of eukaryotic TSPO homologues.

<p>(A) TSPO topology in the membrane. (B) Multiple sequence alignment of eukaryotic TSPO homologues chosen for the initial expression test was performed using Jalview version 2.8.1.</p

Stability of TSPO in detergents.

<p>Stability of TSPO in detergents.</p