Summary We describe the first NMR structure of a polytopic helical membrane protein. DsbB, a bacterial cytoplasmic membrane protein, plays a key role in disulfide bond formation. It re-oxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. We determined the structure of an inter-loop disulfide bond form of DsbB, an intermediate in catalysis. Analysis of the structure and interactions with substrates DsbA and quinone reveals functionally relevant changes induced by these substrates. Analysis of the structure, dynamics measurements, and NMR chemical shifts around the inter-loop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated. Our results demonstrate the extraordinary utility of NMR for functional characterization of polytopic integral membrane proteins and provide novel insights into the mechanism of DsbB catalysis.

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The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most Nterminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.structure ͉ dynamics ͉ membrane fusion ͉ SNARE proteins ͉ NMR S ynaptic release of neurotransmitter requires the docking and fusion of synaptic vesicles at the presynaptic membrane. The fusion reaction is thought to be mediated by the formation of the SNARE complex from its components syntaxin-1a and SNAP-25 in the target membrane and synaptobrevin-2 (Syb) in the vesicle membrane (1). These three proteins together form a complex that consists of a thermally very stable coiled-coil four-helix bundle, as revealed by the crystal structure of the soluble SNARE core complex (2). SNAP-25 contributes two helices to this complex and syntaxin and synaptobrevin each contribute one. More recently, the structure of the postfusion cis-SNARE complex with its C-terminal transmembrane (TM) domain extensions was solved at 3.4 Å resolution (3). Most interestingly, the helices continue through the juxtamembrane linker region and into the membrane, suggesting that force could be transmitted through this region into the membrane leading to membrane bending and eventually membrane fusion. This interesting finding immediately raises the question: how exactly is the assembly and folding of the four-helix bundle coupled into the membrane and how does the energy derived from this reaction ultimately fuse two different membranes into one? A common notion is that an initial trans-SNARE complex forms by pairing vesicle and target membrane SNAREs from their Nterminal ends and progressively folds in a zipper-like fashion toward the C-terminal TM domains. This reaction is thought to pull the two membranes into closer contact until, at some stage they merge into a single membrane. However, structural data on a trans-SNARE complex do not yet exist. It is also not yet known whether zipperfolding of trans-SNARE complexes progresses smoothly and continuously into the membrane or whether this reaction is discontinuous and segmented in some fashion.To understand how the SNARE complex is formed and to find possible reaction intermediates, several structural studies of SNARE proteins in isolation or in binary comp...

Summary Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca2+-sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance (DEER) and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca2+. However, the favored orientation is one where the Ca2+-binding loops are oriented in opposite directions. A similar approach was taken for membrane associated C2A–C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca2+ and membrane binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca2+-dependent manner.