Integrated Structural Biology for Understanding Membrane Proteins: A TSPO Story

Abstract

Structure elucidation for large membrane proteins remains a challenging task. As membrane protein structure determination is entering another stage of probing the ensemble of dynamic structures, methods such as NMR, EPR and EM were used to provide orthogonal information of the structure. We attempted integration of experimental data from multiple sources to allow for structural characterization of IMP structure. We have constructed a pipeline to utilize multiple experimental restraints to fold membrane protein in BCL and Rosetta. The pipeline described in the manuscript could determine structure to an accuracy of 1.2Å RMSD to the experimentally determined structural model. With the same principle of integrating experimental data with computational modeling, I characterized an integral membrane protein (IMPs): The translocator protein (TSPO) and its ligand interactions, as well as a class-C GPCR. TSPO, previously named peripheral-benzodiazepin receptor (PBR) is a membrane protein located on the outer-mitochondrial membrane. It has been proposed to be an important modulator for cholesterol transport, oxidative stress, apoptosis and immune responses. Structural models of mice TSPO (mTSPO) and its homologue from bacterial species determined using different experimental method introduced several controversies. Protein structure exists in a dynamic conformational equilibrium. The experimental system used to determine TSPO structures have their respective perturbations. We experimentally determined that the detergent system to solubilize TSPO affect its ligand binding characteristics. This calls for a further investigation on the membrane systems used to study TSPO. To hopefully resolve some of the controversies in the structural features of TSPO, a computational remodel of mTSPO was constructed. The remodeled mTSPO shares the structural features observed in sequence-conserved region of the protein and agrees with some of the experimental data. We also report the imaging ligand VUIIS8310 binding site on mTSPO based on computational ligand docking to remodeled mTSPO and NMR data.