Synaptic vesicle exocytosis is mediated by the vesicular Ca2+-sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and with acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using both PC12 cells from Rattus norvegicus and artificial supported bilayers we now show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ via PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine (PS) in the vesicle membrane upon Ca2+-triggering. We determined the crystal structure of the C2B-domain of synaptotagmin-1 bound to phosphoserine, allowing for developing a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+-influx bringing the vesicle membrane close enough for membrane fusion.

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

The aggregation and organization of membrane proteins and transmembrane peptides is related to the interacting molecular species itself and strongly depends on the lipid environment. Because of the complexity and dynamics of these interactions, they are often hardly traceable and nearly impossible to predict. For this reason, peptide model systems are a valuable tool in studying membrane associated processes since they are synthetically accessible and can be readily modified. To control and study the aggregation of peptide transmembrane domains (TMDs) the interacting interfaces of the TMDs themselves can be altered. A second less extensively studied approach targets the TMD assembly by using interaction and recognition of domains at the membrane outside as frequently found in the membrane protein interplay and protein assembly. In the present study, double helical transmembrane domains were designed and synthesized on the basis of a recently reported d,l-alternating peptide pore motif derived from gramicidin A. The highly hydrophobic and aromatic transmembrane peptide was covalently functionalized with a short peptide nucleic acid (PNA) used as specific outer-membrane recognition unit. The PNA sequences were chosen with high polarity to ensure localization within the aqueous phase. To estimate the impact of the membrane adjacent recognition on the TMD assembly by Förster resonance energy transfer (FRET), fluorescence probes were covalently attached to the side chains of the membrane spanning peptide helices. Dimerization of the TMD-peptide/PNA conjugates within unilamellar lipid vesicles was observed. The dimer/monomer ratio of TMDs can be controlled by temperature variation.

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as quadruplex forming RNA/PNA oligomers based on fluorescence intensity.