Conformation of the synaptobrevin transmembrane domain

Conformation of the synaptobrevin transmembrane domain
Published online before print May 18, 2006
Mark Bowen*, and Axel T. Brunger
The Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology and Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, CA 94305-5489
The synaptic vesicle protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) forms part of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, which is essential for vesicle fusion. Additionally, the synaptobrevin transmembrane domain can promote lipid mixing independently of complex formation. Here, the conformation of the transmembrane domain was studied by using circular dichroism and attenuated total reflection Fourier-transform infrared spectroscopy. The synaptobrevin transmembrane domain has an -helical structure that breaks in the juxtamembrane region, leaving the cytoplasmic domain unstructured. In phospholipid bilayers, infrared dichroism data indicate that the transmembrane domain adopts a 36? angle with respect to the membrane normal, similar to that reported for viral fusion peptides. A conserved aromatic/basic motif in the juxtamembrane region may be causing this relatively high insertion angle.
Fourier-transform infrared spectroscopy | membrane fusion | membrane protein | neuotransmission
In neurons, synaptic vesicles dock at the plasma membrane and fuse upon an increase in local Ca2+ concentration, releasing neurotransmitters into the synaptic cleft (1?3). Many of the proteins involved in this complex reaction have been identified (4?8). Members of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) family are involved in the final stages of neurotransmitter release. The binding of the synaptic vesicle protein synaptobrevin to the plasma membrane proteins syntaxin and SNAP-25 is essential for stimulated neurotransmitter release. Although the required factors and precise sequence of events that trigger Ca2+-dependent vesicle fusion is hotly debated, the SNAREs are well established as a fundamental yet incomplete part of the fusion machinery (6).
The neuronal SNARE proteins are largely unstructured as monomers (9?12) and undergo a dramatic structural transition to form an -helical heterotrimer (13, 14). The energy from this structural transition was proposed to drive membrane fusion (10, 13, 15), but recent studies have shown that SNARE complex formation can occur without inducing fusion (16, 17) and that SNAREs disrupt membranes and promote lipid mixing independent of complex formation (16, 18?20). Thus, the relationship between structure induction, complex formation, and membrane fusion activity remains unclear.
Synaptobrevin (also called VAMP, vesicle-associated membrane protein) contains a single SNARE motif involved in formation of the complex with syntaxin and SNAP-25 and a C-terminal transmembrane domain. The conserved juxtamembrane region (residues 77?90) binds phospholipids and calmodulin (21). Tryptophan residues in the juxtamembrane region cause portions of the SNARE motif to insert into the bilayer (22). These interactions have been suggested to regulate SNARE complex formation and play a role in vesicle fusion (23?25). However, the effect of the transmembrane domain on the structure of the cytosolic domain has been unknown.
Here we used circular dichroism (CD) and attenuated total reflection (ATR) Fourier-transform infrared (IR) spectroscopy (ATR-FTIR) to examine the structure of synaptobrevin in both detergent and phospholipid environments. ATR-FTIR is a well established method for determining the secondary structure and orientation of transmembrane domains (26). The frequency of the amide I absorption depends on the secondary structure, whereas peptide orientation is related to the observed dichroism (27, 28). In addition, site-specific isotopic labeling with 13C shifts the frequency of the vibrational mode, allowing individual residues to be directly probed (29?32).
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