OPA1 helical structures give perspective to mitochondrial dysfunction.

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria2. Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes3. Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains-a hallmark of the dynamin superfamily of proteins4. OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions.

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions.

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca2+ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb2+, a structural and functional surrogate of Ca2+, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb2+-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, "prime" Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.

Phosphatidylinositol 4,5 Bisphosphate Controls the cis and trans Interactions of Synaptotagmin 1.

Synaptotagmin 1 acts as the Ca2+ sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here, we describe measurements on full-length membrane-reconstituted synaptotagmin 1 using site-directed spin labeling in which we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate, even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+ binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS-containing bilayers and will bind in a cis configuration to membranes containing PS even if a phosphatidylinositol 4,5 bisphosphate membrane is presented in trans. The data are consistent with a bridging activity for synaptotagmin 1 in which the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

The Fluidity of Phosphocholine and Maltoside Micelles and the Effect of CHAPS.

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.e., fluidity). The nitroxide probe rotational dynamics in longer chain detergents is more restricted than in shorter chain detergents, and maltoside micelles are more restricted than phosphocholine micelles. Furthermore, the micelle microviscosity can be modulated with mixtures, as demonstrated with mixtures of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate with n-dodecylphosphocholine, n-tetradecylphosphocholine, n-decyl-ß-D-maltoside, or n-dodecyl-ß-D-maltoside. These results indicate that observed differences in membrane protein stability in these detergents could be due to fluidity in addition to the already determined structural differences.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.

Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus.

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact.

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1.

Synaptotagmin-1 (Syt1) functions as the Ca2+ sensor in neuronal exocytosis, and it is routinely incorporated into lipid bilayers along with other components of the fusion machinery in order to reconstruct the in vivo fusion process. Here, we demonstrate that the detergent used to reconstitute full-length Syt1 has a significant effect on the state of the protein in bilayers. When octyl-ß-d-glucopyranoside is used to reconstitute the protein, Syt1 is present in an aggregated state that is mediated by the long juxta-membrane linker. EPR spectra from spin labels in the two C2 domains of Syt1 no longer resemble those obtained from a soluble construct containing these domains, and the C2B domain no longer exhibits a Ca2+ -dependent membrane insertion. In contrast, when reconstituted using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Syt1 is largely monomeric and the EPR spectra from C2A and C2B resemble those of the soluble construct. This result demonstrates that the choice of detergent used to reconstitute Syt1 can modulate the state of the neuronal Ca2+ -sensor.

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis.

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.

PtdInsP2 and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium.

The Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to PtdIns(4,5)P2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.