Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicleassociated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.clathrin-mediated endocytosis | structure N eurotransmission in the brain depends on the calcium-triggered fusion and recycling of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic membrane at active zones (1). Following their exocytic insertion into the presynaptic membrane, SV proteins need to be retrieved at a precisely defined stoichiometry by endocytosis, a process involving clathrin, adaptors, and other endocytic proteins (2). Fast calcium-triggered SV fusion critically depends on the SV arginine (R)-soluble NSF attachment protein receptor (SNARE) synaptobrevin [or vesicle-associated membrane protein (VAMP)], which by forming a complex with the plasma membrane glutamine (Q)-SNAREs syntaxin and synaptosomal-associated protein (SNAP)-25 (3) drives neuroexocytosis (4, 5). Synapses lacking synaptobrevin 2 display <1% of wild-type release when stimulated by action potential (AP)-mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exoendocytically sorted with very high precision (8). Similar observations have been made for other SV proteins, including synaptotagmin and vesicular glutamate transporters (VGLUTs). How such precise sorting of synaptobrevin 2 is achieved has remained enigmatic. Synaptobrevin lacks recognizable linear sorting motifs (9), and unlike other SNARE proteins does not contain a folded N-terminal domain that serves as a targeting determinant in other VAMP family members (10-12).Genetic data have linked synaptobrevin sorting to the function of the AP180 N-terminal homology (...

Understanding the relationship between protein sequence and structure is one of the great challenges in biology. In the case of the ubiquitous coiled-coil motif, structure and occurrence have been described in extensive detail, but there is a lack of insight into the rules that govern oligomerization, i.e. how many α-helices form a given coiled coil. To shed new light on the formation of two- and three-stranded coiled coils, we developed a machine learning approach to identify rules in the form of weighted amino acid patterns. These rules form the basis of our classification tool, PrOCoil, which also visualizes the contribution of each individual amino acid to the overall oligomeric tendency of a given coiled-coil sequence. We discovered that sequence positions previously thought irrelevant to direct coiled-coil interaction have an undeniable impact on stoichiometry. Our rules also demystify the oligomerization behavior of the yeast transcription factor GCN4, which can now be described as a hybrid—part dimer and part trimer—with both theoretical and experimental justification.

The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537 mBMAL1 acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1.In mammals many physiological processes are regulated in a day-time-dependent manner. These circadian (24 h) rhythms are generated by circadian clocks, which are operated by transcriptional and translational feedback loops. In the central feedback loop, the bHLH-PAS (basic Helix-Loop-Helix-PER-ARNT-SIM) transcription factors mBMAL1 (brain and muscle ARNT-like protein) and mCLOCK (circadian locomotor output cycle kaput) activate the transcription of three period genes (mper1,2,3) and two cryptochromes (mcry1,2) (1). The mPER proteins and (even more potently) the mCRY proteins feedback-repress their own transcription by regulating the activity of mBMAL1 and mCLOCK (2, 3). Notably, the mBMAL1-mCLOCK transcription factor complex not only regulates the mper and mcry genes but also a large number of clock controlled genes, including genes involved in cell cycle regulation, cellular detoxification, and metabolism (4). Hence, the regulation of these transcription factors is of relevance for many body functions and associated diseases (e.g. sleep and depressive disorders, metabolic syndrome, cardiovascular diseases, and tumor formation) that are under the control of the circadian clock (5). The importance of mBMAL1 for clock function is clearly demonstrated by the fact that mBMAL1 Ϫ/Ϫ knock-out mice show an immediate and complete loss of circadian rhythmicity at a behavio...

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.

Two is better than one: Coiled‐coil sequence motifs are versatile protein–protein interaction modules. Single and double substitutions of the GCN4 coiled coil (1, 2, and 3) showed that the core positions a and d in the third heptad act as a switch for the transition from the native homodimeric to homotrimeric structures. This creates a synthetic three‐membered association network with monomeric, homotrimeric, and heterodimeric coiled‐coil states.