Shelagh Ferguson-Miller

The ancient membrane protein TSPO is phylogenetically widespread from archaea and bacteria to insects, vertebrates, plants, and fungi. TSPO’s primary amino acid sequence is only modestly conserved between diverse species, although its five transmembrane helical structure appears mainly conserved. Its cellular location and orientation in membranes have been reported to vary between species and tissues, with implications for potential diverse binding partners and function. Most TSPO functions relate to stress-induced changes in metabolism, but in many cases it is unclear how TSPO itself functions—whether as a receptor, a sensor, a transporter, or a translocator. Much evidence suggests that TSPO acts indirectly by association with various protein binding partners or with endogenous or exogenous ligands. In this review, we focus on proteins that have most commonly been invoked as TSPO binding partners. We suggest that TSPO was originally a bacterial receptor/stress sensor associated with porphyrin binding as its most ancestral function and that it later developed additional stress-related roles in eukaryotes as its ability to bind new partners evolved.

The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is of longstanding medical interest as both a biomarker for neuroinjury and a potential drug target for neuroinflammation and other disorders. Recently it was shown that ligand residence time is a key factor determining steroidogenic efficacy of TSPO-binding compounds. This spurs interest in simulations of (un)binding pathways of TSPO ligands, which could reveal the molecular interactions governing ligand residence time. In this study, we use a weighted ensemble algorithm to determine the unbinding pathway for different poses of PK-11195, a TSPO ligand used in neuroimaging. In contrast with previous studies, our results show that PK-11195 does not dissociate directly into the solvent but instead dissociates via the lipid membrane by going between the transmembrane helices. We analyze this path ensemble in detail, constructing descriptors that can facilitate a general understanding of membrane-mediated ligand binding. We construct a Markov state model using additional straightforward simulations to determine pose stability and kinetics of ligand unbinding. Together we combine over 40µs of trajectory data to form a coherent picture of the ligand binding landscape. We find that all poses are able to interconvert before unbinding, leading to single mean first passage time estimate for all starting poses which roughly agrees with the experimental quantity. The ligand binding transition state predicted by our combined model occurs when PK-11195 is already in the membrane and does not involve direct ligand-protein interactions. This has implications for the design of new long residence-time TSPO ligands.SIGNIFICANCE: Kinetics-oriented drug design is an emerging objective in drug discovery. However, while ligand binding affinity (or the binding free energy) is purely a function of the bound and unbound states, the binding kinetics depends on the nature of the paths by which the (un)binding occurs. This underscores the importance of approaches that can reveal information about the ensemble of (un)binding paths. Here we used advanced molecular dynamics approaches to study the unbinding of PK-11195 from TSPO and find it dissociates from the protein by dissolving into the membrane, and that the transition state occurs after the PK-11195 molecule has already separated from TSPO. These results motivate the design of future long-residence time TSPO ligands that destabilize the membrane-solvated transition state.

Given the central role of cytochrome c oxidase (CcO) in health and disease, it is an increasingly important question as to how the activity and efficiency of this key enzyme are regulated to respond to a variety of metabolic states. The present paper summarizes evidence for two modes of regulation of activity: first, by redox-induced conformational changes involving the K-proton uptake path; and secondly, by ligand binding to a conserved site immediately adjacent to the entrance of the K-path that leads to the active site. Both these phenomena highlight the importance of the K-path in control of CcO. The redox-induced structural changes are seen in both the two-subunit and a new four-subunit crystal structure of bacterial CcO and suggest a gating mechanism to control access of protons to the active site. A conserved ligand-binding site, first discovered as a bile salt/steroid site in bacterial and mammalian oxidases, is observed to bind an array of ligands, including nucleotides, detergents, and other amphipathic molecules. Highly variable effects on activity, seen for these ligands and mutations at the K-path entrance, can be explained by differing abilities to inhibit or stimulate K-path proton uptake by preventing or allowing water organization. A new mutant form in which the K-path is blocked by substituting the conserved carboxyl with a tryptophan clarifies the singularity of the K-path entrance site. Further study in eukaryotic systems will determine the physiological significance and pharmacological potential of ligand binding and conformational change in CcO.

Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer’s and Parkinson’s disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.