Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2.Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1.Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected.Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors.

Synaptotagmin-1 is the calcium sensor for neuronal exocytosis, but the mechanism by which it triggers membrane fusion is not fully understood. Here we show that synaptotagmin accelerates SNARE-dependent fusion of liposomes by interacting with neuronal Q-SNARES in a Ca 2+ -independent manner. Ca 2+ -dependent binding of synaptotagmin to its own membrane impedes the activation. Preventing this cis interaction allows Ca 2+ to trigger synaptotagmin binding in trans, accelerating fusion. However, when an activated SNARE acceptor complex is used, synaptotagmin has no effect on fusion kinetics, suggesting that synaptotagmin operates upstream of SNARE assembly in this system. Our results resolve major discrepancies concerning the effects of full-length synaptotagmin and its C2AB fragment on liposome fusion and shed new light on the interactions of synaptotagmin with SNAREs and membranes. However, our findings also show that the action of synaptotagmin on the fusionarrested state of docked vesicles in vivo is not fully reproduced in vitro.Neurotransmitters are stored in synaptic vesicles that undergo Ca 2+ -dependent exocytosis upon stimulation. Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins, including synaptobrevin-2 (also referred to as VAMP2) in the membrane of synaptic vesicles and syntaxin-1 and SNAP-25 in the plasma membrane. SNAREs are characterized by conserved stretches of 60-70 amino acid residues, referred to as SNARE motifs. Syntaxin-1 and synaptobrevin-2 each possess a single SNARE motif adjacent to the C-terminal transmembrane domain, whereas SNAP-25 contains two SNARE motifs that are separated by a palmitoylated linker 1,2 . The SNARE motifs are unstructured as monomers 3 but assemble into a tight bundle of four a-helices 4 . SNARE motifs are divided into four conserved subfamilies, referred to as Qa-, Qb-, Qc-and R-SNARE motifs. Each SNARE complex contains one member of each subfamily 5 . The assembly of SNARE complexes is currently believed to be the essential reaction in driving membrane fusion. According to this model, the formation of the SNARE complex is initiated in a trans configuration at the N-terminal ends of the SNARE motifs, forming a bridge between the membranes. Assembly then proceeds toward the C-terminal membrane anchor domains, clamping the membranes together and thus overcoming the energy barrier for fusion [6][7][8] .In contrast to several other SNARE-dependent fusion reactions, neuronal exocytosis is strongly upregulated by calcium 9 . The fast component of Ca 2+ -dependent release, which is essential for synchronous, action potential-coupled release, is mediated by the proteins synaptotagmin-1, synaptotagmin-2 and probably synaptotagmin-9, which reside in the membrane of synaptic vesicles 2 . Synaptotagmins constitute a family of type I membrane proteins with widespread tissue distribution 10 . The cytoplasmic part of the synaptotagmins contains t...

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P 2 binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P 2 -enriched plasma membrane.Calcium-dependent secretion of neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission. The underlying membrane fusion reaction between vesicle and plasma membrane has been intensively studied and found to be promoted by both protein-protein as well as protein-lipid interactions. From the multitude of proteins involved in this membrane fusion event, the Ca 2ϩ -binding protein synaptotagmin 1 is one of its central regulating factors (for review, see Refs. 1-6). Synaptotagmin 1 is anchored in the membrane of synaptic vesicles via a single transmembrane region. Its N-terminal region comprises a short luminal domain, whereas the larger cytoplasmic C-terminal region consists of tandem C2 domains, termed C2A and C2B, tethered to each other via a short linker (7) (a schematic outline of the structural features of synaptotagmin 1 is given in Fig. 1A). Several isoforms with similar domain structure have been identified (8).C2 domains are Ca 2ϩ binding modules of ϳ130 amino acids, first described as the second conserved region of protein kinase C (PKC) 2 (9). The C2A domain of synaptotagmin 1 was the first C2 domain structure to be determined (10). In subsequent studies other C2 domains, including the C2B domain of synaptotagmin, were shown to exhibit very similar three-dimensional structures. They have a conserved eight-stranded anti-parallel ␤-sandwich connected by surface loops. C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction (PKC, phospholipases, phosphatidylinositol 3-kinases, etc.) and proteins involved in membrane trafficking (synaptotagmins, rabphilin, DOC2, etc.) (11).Calcium ions bind in a cup-shaped depression formed by the N-and C-terminal loops of the C2 key motifs of C2 domains. Nota...

Neuronal and axonal damage is considered to be the main cause for long-term disability in multiple sclerosis. We analyzed the mechanism and kinetics of neuronal cell death in experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) by combining an electrophysiological in vivo assessment of the optic pathway with the investigation of retinal ganglion cell (RGC) counts. In accordance with our previous findings in this animal model, neuritis of the optic nerve (ON) leads to apoptotic RGC death. By further investigating the time course of RGC apoptosis in the present study, we found that neuronal cell death together with decreased visual acuity values occurred before the onset of clinical symptoms. Simultaneously with the time course of RGC apoptosis, we found a down-regulation of phospho-Akt as well as a shift in the relation of 2 proteins of the Bcl-2 family, Bax and Bcl-2, towards a more proapoptotic ratio in these cells. Comparing the kinetics and mechanisms of RGC death during MOG-EAE with those following complete surgical transection of the ON, we found significant agreement. We hypothesize that the main reason for RGC loss in MOG-EAE is the inflammatory attack but RGC death also occurs independently of histopathological ON changes.

Fibroblasts exhibit heterogeneous cell geometries in tissues and integrate both mechanical and biochemical signals in their local microenvironment to regulate genomic programs via chromatin remodelling. While in connective tissues fibroblasts experience tensile and compressive forces (CFs), the role of compressive forces in regulating cell behavior and, in particular, the impact of cell geometry in modulating transcriptional response to such extrinsic mechanical forces is unclear. Here we show that CF on geometrically well-defined mouse fibroblast cells reduces actomyosin contractility and shuttles histone deacetylase 3 (HDAC3) into the nucleus. HDAC3 then triggers an increase in the heterochromatin content by initiating removal of acetylation marks on the histone tails. This suggests that, in response to CF, fibroblasts condense their chromatin and enter into a transcriptionally less active and quiescent states as also revealed by transcriptome analysis. On removal of CF, the alteration in chromatin condensation was reversed. We also present a quantitative model linking CF-dependent changes in actomyosin contractility leading to chromatin condensation. Further, transcriptome analysis also revealed that the transcriptional response of cells to CF was geometry dependent. Collectively, our results suggest that CFs induce chromatin condensation and geometry-dependent differential transcriptional response in fibroblasts that allows maintenance of tissue homeostasis.