Mitochondrial homeostasis is tightly controlled by ubiquitination. The mitochondrial integral membrane ubiquitin ligase MARCH5 is a crucial regulator of mitochondrial membrane fission, fusion, and disposal through mitophagy. In addition, the lipid composition of mitochondrial membranes can determine mitochondrial dynamics and organelle turnover. However, how lipids influence the ubiquitination processes that control mitochondrial homeostasis remains unknown. Here, we show that lipids common to the mitochondrial membranes interact with MARCH5 and affect its activity and stability depending on the lipid composition in vitro. As the only one of the tested lipids, cardiolipin binding to purified MARCH5 induces a significant decrease in thermal stability, whereas stabilisation increases the strongest in the presence of phosphatidic acid. Furthermore, we observe that the addition of lipids to purified MARCH5 alters the ubiquitination pattern. Specifically, cardiolipin enhances auto-ubiquitination of MARCH5. Our work shows that lipids can directly affect the activity of ubiquitin ligases and suggests that the lipid composition in mitochondrial membranes could control ubiquitination-dependent mechanisms that regulate the dynamics and turnover of mitochondria.
Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.
Abstract α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α 1B AR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α 1B AR structure allows the identification of two unique secondary binding pockets. By structural comparison of α 1B AR with α 2 ARs, and by constructing α 1B AR-α 2C AR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α 1B AR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the membrane and interacting with membrane-embedded proteins to alternate their activity and stability. Like lipids, membrane proteins are amphipathic and are stabilized by the hydrophobic forces of the lipid bilayer. Membrane protein-lipid interactions are difficult to investigate since membrane proteins need to be reconstituted in a lipid-mimicking environment. A common and well-established approach is the detergent-based solubilization of the membrane proteins in detergent micelles. Nowadays, nanodiscs and liposomes are used to mimic the lipid bilayer and enable the work with membrane proteins in a more natural environment. However, these protocols need optimization and are labor intensive. The present protocol describes straightforward instructions on how the preparation of lipids is performed and how the lipid detergent mixture is integrated with the membrane protein MARCH5. The lipidation protocol was performed prior to an activity assay specific to membrane-bound E3 ubiquitin ligases and a stability assay that could be used for any membrane protein of choice.
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