BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). Indeed, BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand binding. To better understand the conformation and dynamics of this key region of BCL-XL we have developed methods for isolating the membrane-embedded C-terminal tail from its N-terminal head and for preparing protein suitable for structural and biochemical studies. Here, we outline the methods for sample preparation and characterization and describe previously unreported structural and dynamics features. We show that the C-terminal tail of BCL-XL forms a transmembrane α-helix that retains a significant degree of conformational dynamics. We also show that the presence of the intact C-terminus destabilizes the soluble state of the protein, and that the small fraction of soluble recombinant protein produced in E. coli is susceptible to proteolytic degradation of C-terminal residues beyond M218. This finding impacts the numerous previous studies where recombinant soluble BCL-XL was presumed to be full-length. Notably, however, the majority of recombinant BCL-XL produced in E. coli is insoluble and protected from proteolysis. This protein retains the complete C-terminal tail and can be reconstituted in lipid bilayers in a folded and active state.

The interactions of Bcl-2 family proteins with intracellular lipids are essential for the regulation of apoptosis, a mechanism of programmed cell death that is central to the health and development of multicellular organisms. Bid and its caspase-8 cleavage product, tBid, promote the permeabilization of the mitochondrial outer membrane and sequester antiapoptotic Bcl-2 proteins to counter their cytoprotective activity. Bid and tBid also promote lipid exchange, a characteristic trait of apoptosis. Here, we show that tBid is capable of associating with phospholipids to form soluble, nanometer-sized lipoprotein particles that retain binding affinity for the antiapoptotic protein Bcl-xL. The tBid lipoprotein particles form with a lipid/protein stoichiometry in the range of 20/1 and have a diameter of $11.5 nm. Lipoparticle-bound tBid retains an a-helical structure and binds Bcl-xL through its third Bcl-2 homology motif, forming a soluble, lipid-associated heteroprotein complex. The results shed light on the role of lipids in mediating Bcl-2 protein mobility and interactions.

Phosphatidylinositol is critical for intracellular signaling and anchoring of carbohydrates and proteins to outer cellular membranes. In eukaryotes, phosphatidylinositol-based lipids play important roles in numerous aspects of signal transduction and in the anchoring of glycosylphosphatidylinositol (GPI) linked proteins to the membrane. In prokaryotes, phosphatidylinositol (PI) is produced by mycobacteria, as well as some other bacterial genera, where it is required for the biosynthesis of key components of the cell wall, such as the glycolipids lipomannan and lipoarabinomannan, which are tethered to the membrane via a common PI anchor. In Mycobacterium tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response. The defining step in phosphatidylinositol biosynthesis is catalyzed by CDPalcohol phosphotransferases (CD-APs), transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. In prokaryotes, this reaction is catalyzed by the CDP-AP phosphatidylinositol-phosphate synthase (PIPS) to yield phosphatidylinositol-phosphate, which is in turn dephosphorylated by an as yet uncharacterized enzyme to PI. Given its essentiality in mycobacterial viability-conditional knockouts prove fatal-and its divergence from the eukaryotic counterpart, PIPS can be considered a promising target for anti-tuberculosis drugs. We have determined structures of PIPS from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization and mutagenesis of the 40%-identical PIPS orthologfrom Mycobacterium tuberculosis supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.