Abstract
The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol–binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N–terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C–terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150–X–Y152–X(3)–R156 and R135–X(2)–Y138–X(2)–L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC–resembling motif that we observed in TM I (residues L17–X(2)–F20–X(3)–R24) and to CRAC in TM V. We expect that the CRAC–like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.
Highlights
The translocator protein (TSPO) is a transmembrane protein (18kDa), evolutionary conserved and expressed in different organisms, from bacteria to humans [1]
Cholesterol interacts almost twice more frequently with the lower part of the helix that is in the inner membrane leaflet than with the upper part of the helix that is in the outer membrane leaflet
Since apo transmembrane helices (TMs) II binds more cholesterol than holo, it is clear that cholesterol has higher binding affinity for the lower part of this helix
Summary
The translocator protein (TSPO) is a transmembrane protein (18kDa), evolutionary conserved and expressed in different organisms, from bacteria to humans [1]. Its biological functions are conserved throughout the phylogenetic spectrum, like tetrapyrrole biosynthesis and/or sterol metabolism [1,2]. The bacterial TSPO homology in Rhodobacter sphaeroides can be functionally replaced by rat TSPO [3], despite that these proteins share only about 30% sequence identity. The human protein (hTSPO) is expressed in all tissues and located in the outer mitochondrial membrane [4,5]. Its highest expression levels are found in steroid-synthesizing cells of endocrine organs indicating that it may play an important role in steroid synthesis from cholesterol [6]. Mammalian TSPO binds cholesterol with high affinity by the cholesterol recognition/interaction amino acid consensus (CRAC)
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