Construction and validation of an atomic model for bacterial TSPO from electron microscopy density, evolutionary constraints, and biochemical and biophysical data

Abstract

The 18 kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications.

Lost in translocation: the functions of the 18-kD translocator protein

“…Because of the high affinity of the cholesterol-TSPO interaction, an additional, currently unknown, mechanism would then be required to release cholesterol. Potential subsequent transport/translocation of cholesterol might occur at a TSPO oligomerization interface [35,36,38,44,45], in agreement with the ability of TSPO to form polymers in vivo [46].…”

Structure of the mammalian TSPO/PBR protein

“…Because TM helix 1 is the least conserved of the five TM regions and is more polar in RsTspO than in mTSPO/PBR, where the functional unit is a monomer, we suggested that TM helix 1 is involved in the dimer interface of RsTspO [22]. However, alternative models were also proposed [31,32].…”

Applications of sequence coevolution in membrane protein biochemistry

“…This method would have helped resolve the L6 loop of the β-barrel protein FhaC in the original crystal structure [74,75], as explained above. In a different study the mitochondrial translocator protein TSPO dimer structure was disputed based on differences in data from NMR [126], EM of helical crystals [127], Rosetta modeling [128], and sequence coevolution analysis [40,129]. A high resolution lipidic cubic phase crystal structure finally resolved the structure of the TSPO dimer [130], which had the topology predicted from sequence coevolution analysis.…”