Abstract
Glycosphingolipids are minor but essential components of cell membrane that exhibit important biological function in membrane trafficking and cell signaling. These glycolipids however are highly diverse in their carbohydrate head group size, composition and their ceramide chain length. For glycolipids with big and complex head group such as GM1, their head group is far more extended out from choline head of PC, providing an efficient contact with proteins. However for some glycolipids such GalCer, GluCer, and sulfatide (SGC), their head group size is of similar size or even smaller than choline of PC (major component of cell membrane). How proteins can efficiently interact with these glycolipids remains to be illustrated. By using solution and solid state NMR, herein we provide evidence suggesting a protruding state of sulfatide head group. We identified the intermolecular NOEs between protons at interface region of SGC and proton of choline head of DPC micelles. Similar intermolecular NOEs also evidences for SGC/LPC and GM1/DPC micelle. To rule out possibilities that these glycolipids protruding out from PC micelle due to high membrane curvature of micelles, we further perform HRMAS experiments on fully hydrated SGC/POPC with or without cholesterol at multilamellar condition, showing that at mixing time of 50ms ~ 600ms, similar intermolecular NOEs can also be observed. Along with X-ray diffraction data that gives the membrane thickness information of SGC/POPC multilamellar, we conclude that SGC may dynamically protrude out from PC to provide an efficient mechanism for protein recognition. The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming □-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for the CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pore with pore size and lifetime in the range of about 30 Å and 10-2 sec, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch-clamp electrophysiological experiment on the plasma membrane of H9C2 cells. We also derived the peripheral binding structural model of CTX A3□sulfatide complex in sulfatide containing PC micelles by NMR and molecular docking method and compare with other CTX A3□sulfatide complex structure determined previously by X-ray in membrane-like environment. The NMR results indicate that sulfatide head group conformation changes from a bent shovel (-sc/ap) to an extended (sc/ap) conformation upon initial binding of CTX A3. An additional global reorientation of sulfatide molecule is also needed for CTX A3 dimer formation as inferred by the difference between the X-ray and NMR complex structure. Since the overall folding of CTX A3 molecules remain the same, sulfatide in phospholipids bilayer is proposed to play an active role by involving its local and global conformational changes to promote both the oligomerization and reorientation of CTX A3 molecule for its transient pore formation and cell internalization. Cobra cardiotoxins (CTXs) are three-fingered polypeptides with positively charged domains that have been shown to bind to anionic ligands of snake venom citrate, glycosaminoglycans, sulfoglycosphingolipid and nucleotide triphosphate with various biochemical effects including toxin dimerization, cell surface retention, membrane pore formation, cell internalization and blocking of enzymatic activities of kinase and ATPase. The reported anionic binding sites, however, are found to be different among different CTX homologues for potentially different CTX activities. Herein, by NMR studies of the binding of inorganic phosphate, dATP (stable form of ATP), and heparin-derived tetrasaccharide to Naja atra CTX A1, a novel CTX molecule exhibiting in vivo necrotic activity on skeletal muscle; we demonstrate that diverse ligands binding to CTXs could also occur at a single protein site with flexible side chain interactions. The flexibility of such an interaction is also illustrated by the available heparin□CTX A3 complex structures with different heparin chain lengths binding at the same site. Our results provide a likely structural explanation on how the interaction between heparan sufate and proteins depends more on the overall charge cluster organization rather than on their fine structures. We also suggest that the ligand binding site of CTX homologues can be fine-tuned by nonconserved residues near the binding pocket because of their flexible side chain interaction and dimerization ability, even for the rigid CTX molecules tightened by four disulfide bonds.