Carsten C. Mahrenholz

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicle-associated 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.

Quantitative analyses of cryptochrome - mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock

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-537mBMAL1 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.

A study to assess the cross-reactivity of cellulose membrane-bound peptides with detection systems: an analysis at the amino acid level

The growing demand for binding assays to study protein–protein interaction can be addressed by peptide array‐based methods. The SPOT technique is a widespread peptide‐array technology, which is able to distinguish semi‐quantitatively the binding affinities of peptides to defined protein targets within one array. The quality of an assay system used for probing peptide arrays depends on the well‐balanced combination of screening and read‐out methods. The former address the steady‐state of analyte capture, whereas the latter provide the means to detect captured analyte. In all cases, however, false‐positive results can occur when challenging a peptide array with analyte or detecting captured analyte with label conjugates. Little is known about the cross‐reactivity of peptides with the detection agents. Here, we describe at the amino acid level the potential of (i) 5‐(and 6)‐carboxytetramethylrhodamine (5(6)‐TAMRA), (ii) fluoresceinisothiocyanate in form of the peptide‐bound fluorescein‐substituted thiourea derivative (FITC), and (iii) biotin/streptavidin‐POD to cross‐react with individual amino acids in a peptide sequence. Copyright

Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify.

Investigation of the network of preferred interactions in an artificial coiled-coil association using the peptide array technique

Summary We screened a randomized library and identified natural peptides that bound selectively to a chimeric peptide containing α-, β- and γ-amino acids. The SPOT arrays provide a means for the systematic study of the possible interaction space accessible to the αβγ-chimera. The mutational analysis reveals the dependence of the binding affinities of α-peptides to the αβγ-chimera, on the hydrophobicity and bulkiness of the side chains at the corresponding hydrophobic interface. The stability of the resulting heteroassemblies was further confirmed in solution by CD and thermal denaturation.

PrOCoil – Advances in predicting two- and three-stranded coiled coils

Introduction CCs consist of 2 to 5 alpha-helices that are wrapped around each other with a left-handed super-helical twist. Their ubiquity and the stable interactions of their helices make coiled coils ideal building blocks for designing novel proteins. Furthermore, CC interactions have recently attracted attention as promising drug targets. Abstract The aim of this project was to investigate which specific properties influence the oligomerization of coiled coil (CC) proteins. Based on comprehensive peptide libraries of GCN4 mutants, our results indicated that the formation of oligomeric structures strongly depends on various specific positions of a heptad. To investigate these dependencies, we collected all known dimeric and trimeric CCs in the PDB and applied Support Vector Machines (SVMs) to classify them. We used pairwise feature extraction to identify characteristic amino acid patterns that determine the rules for oligomerization. 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. PrOCoil is also available as a web tool (http://www.bioinf.jku.at/software/procoil/).