Structural Insights into the Binding and Degradation Mechanisms of Protoporphyrin IX by the Translocator Protein TSPO.

The 18 kDa translocator protein (TSPO) has gained considerable attention as a clinical biomarker for neuroinflammation and a potential therapeutic target. However, the mechanisms by which TSPO associates with ligands, particularly the endogenous porphyrin ligand protoporphyrin IX (PpIX), remain poorly understood. In this study, we employed mutagenesis- and spectroscopy-based functional assays to investigate TSPO-mediated photo-oxidative degradation of PpIX and identify key residues involved in the reaction. We provide structural evidence using electron spin resonance, which sheds light on the highly conserved intracellular loop (LP1) connecting transmembrane 1 (TM1) and TM2. Our findings show that LP1 does not act as a lid to regulate ligand binding; instead, it interacts strongly with the TM3-TM4 linker (LP3) to stabilize the local structure of LP3. This LP1-LP3 interaction is crucial for maintaining the binding pocket structure, which is essential for proper ligand binding. Our results also demonstrate that PpIX accesses the pocket through the lipid bilayer without requiring conformational changes in TSPO. This study provides an improved understanding of TSPO-mediated PpIX degradation, highlighting potential therapeutic strategies to regulate the reaction.

Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop.

BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.

Doubly spin-labeled nanodiscs to improve structural determination of membrane proteins by ESR.

Pulsed dipolar spectroscopy (PDS) is a powerful tool to explore conformational changes of membrane proteins (MPs). However, the MPs suffer from relatively weak dipolar signals due to their complex nature in membrane environments, which consequently reduces the interspin distance resolution obtainable by PDS. Here we report the use of nanodiscs (NDs) to improve the distance resolution. Two genetically engineered membrane scaffold protein mutants are introduced, each of which is shown to form double-labeled ND efficiently and with high homogeneity. The resultant interspin distance distribution is featured by a small distribution width, suggesting high resolution. When PDS is performed on a binary mixture of the double-labeled ND devoid of MPs and the un-labeled ND with incorporated double-labeled MPs, the overall amplitude of dipolar signals is increased, leading to a critical enhancement of the distance resolution. A theoretical foundation is provided to validate the analysis. With this approach, the determination of MP structures can be studied at high resolution in NDs.