AfTMEM16/nanodisc complex in the presence of Ca2+
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ABSTRACT:
SUBMITTER: Alessio Accardi
PROVIDER: EMPIAR-10239 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit ?-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis1. Additionally, ?-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of ?-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of ?-arrestin 1 (?arr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-?arr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of ?arr1 to phosphorylated receptor residues and insertion of the finger loop of ?arr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric Go protein complex3. Moreover, the cryo-electron microscopy map reveals that the C-edge of ?arr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, ?arr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of ?-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility.
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Project description:Sarco(endo)plasmic reticulum Ca2+-ATPase catalyzes ATP-driven Ca2+ transport from the cytoplasm to the lumen and is critical for a range of cell functions, including muscle relaxation. Here, we investigated the effects of the headgroups of the 1-palmitoyl-2-oleoyl glycerophospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol (PG) on sarcoplasmic reticulum (SR) Ca2+-ATPase embedded into a nanodisc, a lipid-bilayer construct harboring the specific lipid. We found that Ca2+-ATPase activity in a PC bilayer is comparable with that of SR vesicles and is suppressed in the other phospholipids, especially in PS. Ca2+ affinity at the high-affinity transport sites in PC was similar to that of SR vesicles, but 2-3-fold reduced in PE and PS. Ca2+ on- and off-rates in the non-phosphorylated ATPase were markedly reduced in PS. Rate-limiting phosphoenzyme (EP) conformational transition in 0.1 m KCl was as rapid in PC as in SR vesicles, but slowed in other phospholipids, especially in PS. Using kinetic plots of the logarithm of rate versus the square of mean activity coefficient of solutes in 0.1-1 m KCl, we noted that PC is optimal for the EP transition, but PG and especially PS had markedly unfavorable electrostatic effects, and PE exhibited a strong non-electrostatic restriction. Thus, the major SR membrane lipid PC is optimal for all steps and, unlike the other headgroups, contributes favorable electrostatics and non-electrostatic elements during the EP transition. Our analyses further revealed that the surface charge of the lipid bilayer directly modulates the transition rate.
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Project description:The Na(+)/Ca(2+) exchanger is the major exporter of Ca(2+) across the cell membrane of cardiomyocytes. The activity of the exchanger is regulated by a large intracellular loop that contains two Ca(2+)-binding domains, calcium-binding domain (CBD) 1 and CBD2. CBD1 binds Ca(2+) with much higher affinity than CBD2 and is considered to be the primary Ca(2+) sensor. The effect of Ca(2+) on the structure and dynamics of CBD1 has been characterized by NMR spectroscopy using chemical shifts, residual dipolar couplings, and spin relaxation. Residual dipolar couplings are used in a new way for residue selection in the determination of the anisotropic rotational diffusion tensor from spin relaxation data. The results provide a highly consistent description across these complementary data sets and show that Ca(2+) binding is accompanied by a selective conformational change among the binding site residues. Residues that exhibit a significant conformational change are also sites of altered dynamics. In particular, Ca(2+) binding restricts the mobility of the major acidic segment and affects the dynamics of several nearby binding loops. These observations indicate that Ca(2+) elicits a local transition to a well-ordered coordination geometry in the CBD1-binding site.
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Project description:Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high-resolution structures. Prior to achieving a high-resolution structure, we are investigating whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which are more similar to the native membrane environment than are the widely used detergent micelles. We describe here results that show a protein's outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with FPOP is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topology.
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