Membrane Binding Induces Distinct Structural Signatures in the Mouse Complexin-1C-Terminal Domain.

Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.1.

3-O-Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake.

Prion-like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1-/- (HS 3-O-sulfotransferase-1 knockout) cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau-HS binding and likely the transcellular spread of tau, providing a novel target for disease-modifying treatment of AD and other tauopathies.

Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function.

Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo.

Synaptic vesicles position complexin to block spontaneous fusion.

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its α-helical domain and the SNARE complex. In addition, complexin's C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexin's C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion.

¹H, ¹³C, and ¹5N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4).

Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete (1)H, (15)N, and (13)C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4.

STARD4 abundance regulates sterol transport and sensing.

Nonvesicular transport of cholesterol plays an essential role in the distribution and regulation of cholesterol within cells, but it has been difficult to identify the key intracellular cholesterol transporters. The steroidogenic acute regulatory-related lipid-transfer (START) family of proteins is involved in several pathways of nonvesicular trafficking of sterols. Among them, STARD4 has been shown to increase intracellular cholesteryl ester formation and is controlled at the transcriptional level by sterol levels in cells. We found that STARD4 is very efficient in transporting sterol between membranes in vitro. Cholesterol levels are increased in STARD4-silenced cells, while sterol transport to the endocytic recycling compartment (ERC) and to the endoplasmic reticulum (ER) are enhanced upon STARD4 overexpression. STARD4 silencing attenuates cholesterol-mediated regulation of SREBP-2 activation, while its overexpression amplifies sterol sensing by SCAP/SREBP-2. To analyze STARD4's mode of action, we compared sterol transport mediated by STARD4 with that of a simple sterol carrier, methyl-ß-cyclodextrin (MCD), when STARD4 and MCD were overexpressed or injected into cells. Interestingly, STARD4 and cytosolic MCD act similarly by increasing the rate of transfer of sterol to the ERC and to the ER. Our results suggest that cholesterol transport mediated by STARD4 is an important component of the cholesterol homeostasis regulatory machinery.

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, ß-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type αS and the C-terminal truncation mutant αS1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type αS, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that αS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced αS aggregation both in vitro and in vivo.

The lipid-binding domain of wild type and mutant alpha-synuclein: compactness and interconversion between the broken and extended helix forms.

Alpha-synuclein (alphaS) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits, and by the existence of three alphaS point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function of alphaS is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles, alphaS undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated helices. Membrane-bound conformations of alphaS likely mediate the function of the protein, but may also play a role in the aggregation and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the detergent- and membrane-bound conformations of alphaS, as well as factors that govern the transition of the protein between the extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all three PD-linked alphaS mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition, we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether the protein adopts the broken or extended helix conformation.

Src activates Abl to augment Robo1 expression in order to promote tumor cell migration.

Cell migration is an essential step in cancer invasion and metastasis. A number of orchestrated cellular events involving tyrosine kinases and signaling receptors enable cancer cells to dislodge from primary tumors and colonize elsewhere in the body. For example, activation of the Src and Abl kinases can mediate events that promote tumor cell migration. Also, activation of the Robo1 receptor can induce tumor cell migration. However, while the importance of Src, Abl, and Robo1 in cell migration have been demonstrated, molecular mechanisms by which they collectively influence cell migration have not been clearly elucidated. In addition, little is known about mechanisms that control Robo1 expression. We report here that Src activates Abl to stabilize Robo1 in order to promote cell migration. Inhibition of Abl kinase activity by siRNA or kinase blockers decreased Robo1 protein levels and suppressed the migration of transformed cells. We also provide evidence that Robo1 utilizes Cdc42 and Rac1 GTPases to induce cell migration. In addition, inhibition of Robo1 signaling can suppress transformed cell migration in the face of robust Src and Abl kinase activity. Therefore, inhibitors of Src, Abl, Robo1 and small GTPases may target a coordinated pathway required for tumor cell migration.

E46K Parkinson's-linked mutation enhances C-terminal-to-N-terminal contacts in alpha-synuclein.

Parkinson's disease (PD) is associated with the deposition of fibrillar aggregates of the protein alpha-synuclein (alphaS) in neurons. Intramolecular contacts between the acidic C-terminal tail of alphaS and its N-terminal region have been proposed to regulate alphaS aggregation, and two originally described PD mutations, A30P and A53T, reportedly reduce such contacts. We find that the most recently discovered PD-linked alphaS mutation E46K, which also accelerates the aggregation of the protein, does not interfere with C-terminal-to-N-terminal contacts and instead enhances such contacts. Furthermore, we do not observe a substantial reduction in such contacts in the two previously characterized mutants. Our results suggest that C-terminal-to-N-terminal contacts in alphaS are not strongly protective against aggregation, and that the dominant mechanism by which PD-linked mutations facilitate alphaS aggregation may be altering the physicochemical properties of the protein such as net charge (E46K) and secondary structure propensity (A30P and A53T).

Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.

We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that alphaS does not retain a "hairpin" structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.

Inter-helix distances in lysophospholipid micelle-bound alpha-synuclein from pulsed ESR measurements.

We demonstrate the use of pulsed ESR spectroscopy to measure intramolecular distances in the Parkinson's disease-associated protein alpha-synuclein bound to detergent and lysophospholipid micelles. We show that the inter-helical separation between the two helices formed upon binding to micelles is dependent on micelle composition, with micelles formed from longer acyl chains leading to an increased splaying of the two helices. Our data suggest that the topology of alpha-synuclein is not strongly constrained by the linker region between the two helices and instead depends on the geometry of the surface to which the protein is bound.

Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy.

Alpha-synuclein (alphaS) is a soluble synaptic protein that is the major proteinaceous component of insoluble fibrillar Lewy body deposits that are the hallmark of Parkinson's disease. The interaction of alphaS with synaptic vesicles is thought to be critical both to its normal function as well as to its pathological role in Parkinson's disease. We demonstrate the use of fluorescence correlation spectroscopy as a tool for rapid and quantitative analysis of the binding of alphaS to large unilamellar vesicles of various lipid compositions. We find that alphaS binds preferentially to vesicles containing acidic lipids, and that this interaction can be blocked by increasing the concentration of NaCl in solution. Negative charge is not the only factor determining binding, as we clearly observe binding to vesicles composed entirely of zwitterionic lipids. Additionally, we find enhanced binding to lipids with less bulky headgroups. Quantification of the protein-to-lipid ratio required for binding to different lipid compositions, combined with other data in the literature, yields an upper bound estimate for the number of lipid molecules required to bind each individual molecule of alphaS. Our results demonstrate that fluorescence correlation spectroscopy provides a powerful tool for the quantitative characterization of alphaS-lipid interactions.