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Intrinsically disordered proteins in synaptic vesicle trafficking and release

Open AccessPublished:January 30, 2019DOI:https://doi.org/10.1074/jbc.REV118.006493
      The past few years have resulted in an increased awareness and recognition of the prevalence and roles of intrinsically disordered proteins and protein regions (IDPs and IDRs, respectively) in synaptic vesicle trafficking and exocytosis and in overall synaptic organization. IDPs and IDRs constitute a class of proteins and protein regions that lack stable tertiary structure, but nevertheless retain biological function. Their significance in processes such as cell signaling is now well accepted, but their pervasiveness and importance in other areas of biology are not as widely appreciated. Here, we review the prevalence and functional roles of IDPs and IDRs associated with the release and recycling of synaptic vesicles at nerve terminals, as well as with the architecture of these terminals. We hope to promote awareness, especially among neuroscientists, of the importance of this class of proteins in these critical pathways and structures. The examples discussed illustrate some of the ways in which the structural flexibility conferred by intrinsic protein disorder can be functionally advantageous in the context of cellular trafficking and synaptic function.

      Introduction

      A basic tenet of cell biology is that the eukaryotic cell is compartmentalized into discrete membrane-enclosed organelles and that cellular compartmentalization is critical for biological function. Trafficking between cellular compartments is achieved in part through membrane-enclosed vesicles that bud from a source membrane, travel through the cell, and fuse specifically with a given target membrane. Neurons present interesting and unique challenges for these types of processes. Lengthy axonal and dendritic processes necessitate long-distance trafficking in neurons. In addition, in contrast to the more ubiquitous and continuous nature of vesicular trafficking elsewhere in the cell, synaptic communication between neurons requires highly-localized and precisely-timed release of neurotransmitter into the synaptic cleft through fusion of the synaptic vesicle with the plasma membrane. This process is highly regulated by a number of accessory proteins (
      • Rizo J.
      • Rosenmund C.
      Synaptic vesicle fusion.
      • Rizo J.
      • Südhof T.C.
      The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices–guilty as charged?.
      ,
      • Rizo J.
      • Xu J.
      The synaptic vesicle release machinery.
      ,
      • Südhof T.C.
      • Rizo J.
      Synaptic vesicle exocytosis.
      • Südhof T.C.
      The presynaptic active zone.
      ), some of which are intrinsically disordered or contain functionally critical disordered regions. Accordingly, intrinsically disordered proteins and regions play critical roles in synaptic transmission.
      Protein structure exists as a continuum, with folded, ordered, and well-structured proteins and domains at one extreme and flexible, dynamic, and intrinsically disordered proteins and regions (IDPs
      The abbreviations used are: IDP
      intrinsically disordered protein
      IDR
      intrinsically disordered protein region
      PTM
      post-translational modification
      ER
      endoplasmic reticulum
      GAP
      GTPase-activating protein
      ALPS
      amphipathic lipid-packing sensor
      NSF
      N-ethylmaleimide-sensitive factor
      SNAP
      soluble NSF attachment protein
      NTD
      N-terminal domain
      CTD
      C-terminal domain
      AH
      accessory helix
      CH
      central helix
      SH
      Src homology
      RIM-BP
      RIM-binding protein
      Syt1
      synaptotagmin-1
      MARCKS
      membrane-binding region of the myristoylated alanine-rich protein kinase C substrate
      CaMKII
      calcium/calmodulin-dependent protein kinase II
      PSD
      post-synaptic density
      SV
      synaptic vesicle
      NAC
      non-amyloid-beta-component.
      and IDRs, respectively) at the other (see a recent review by Burger et al. (
      • Burger V.M.
      • Nolasco D.O.
      • Stultz C.M.
      Expanding the range of protein function at the far end of the order-structure continuum.
      ) for a discussion of the term “disordered” versus “unstructured”). This review begins with a brief summary of biophysical properties of IDPs and their interactions before discussing a number of notable examples of how intrinsic disorder contributes to vesicular trafficking in general. We then focus on structural disorder within a number of proteins critical for synaptic function, especially synaptic vesicle release and recycling. Finally, we close with a discussion of the potential role of IDP/IDR-mediated phase transitions and membrane-less organelles in the organization of key elements of the synapse.

      Unique IDP properties confer conformational and functional flexibility

      The primary sequences of IDPs contain a high proportion of charged residues, with few hydrophobic amino acids (
      • Cortese M.S.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in scaffold proteins: getting more from less.
      ,
      • Uversky V.N.
      Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.
      ). Although IDPs feature relatively simple primary sequences, their inability to spontaneously fold into a unique three-dimensional structure leads to great structural complexity. Charge content and patterning within IDP sequences alter the extent of chain collapse, and the sequence composition determines how IDPs respond to external factors like ionic strength and temperature (
      • Shammas S.L.
      • Crabtree M.D.
      • Dahal L.
      • Wicky B.I.
      • Clarke J.
      Insights into coupled folding and binding mechanisms from kinetic studies.
      ). IDPs usually show relatively flat, free energy landscapes, with local minima separated by low barriers, and they tend to rapidly fluctuate between different disordered conformations (
      • Papoian G.A.
      Proteins with weakly funneled energy landscapes challenge the classical structure-function paradigm.
      ).
      Conformational flexibility allows IDPs to interact with other macromolecules in a variety of ways. Indeed, IDPs can be promiscuous binders capable of interacting not only with multiple proteins (which may be other IDP/IDRs or structured proteins/domains), but also with lipid membranes or nucleic acids. IDP interactions often involve folding of the IDP/IDR, but folding upon binding is not an absolute requirement (
      • Uversky V.N.
      Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.
      ,
      • Shammas S.L.
      • Crabtree M.D.
      • Dahal L.
      • Wicky B.I.
      • Clarke J.
      Insights into coupled folding and binding mechanisms from kinetic studies.
      ,
      • Bah A.
      • Forman-Kay J.D.
      Modulation of intrinsically disordered protein function by post-translational modifications.
      ). IDPs are thought to engage their targets through conformational selection or induced fit, although these are not mutually exclusive, and both are likely to occur in different contexts (
      • Arai M.
      • Sugase K.
      • Dyson H.J.
      • Wright P.E.
      Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding.
      ). In conformational selection, some subset of the IDP structural ensemble adopts a conformation appropriate for binding, and the partner subsequently interacts with this preformed structure. In induced fit, binding precedes folding via an initial encounter complex (
      • Cortese M.S.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in scaffold proteins: getting more from less.
      ,
      • Shammas S.L.
      • Crabtree M.D.
      • Dahal L.
      • Wicky B.I.
      • Clarke J.
      Insights into coupled folding and binding mechanisms from kinetic studies.
      ).
      It is not surprising, perhaps, that IDPs often function in signaling networks as hub proteins that integrate multiple signals to link multiple signaling pathways (
      • Cortese M.S.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in scaffold proteins: getting more from less.
      ,
      • Uversky V.N.
      Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.
      ,
      • Bah A.
      • Forman-Kay J.D.
      Modulation of intrinsically disordered protein function by post-translational modifications.
      ). IDP/protein interactions tend to be of low affinity yet high specificity, a feature that is often coupled to regulatory functions within signaling networks: the interactions can be easily and rapidly turned “on” or “off” as required (
      • Cortese M.S.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in scaffold proteins: getting more from less.
      ,
      • Uversky V.N.
      Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.
      • Shammas S.L.
      • Crabtree M.D.
      • Dahal L.
      • Wicky B.I.
      • Clarke J.
      Insights into coupled folding and binding mechanisms from kinetic studies.
      ). In some cases, IDP/IDRs play critical roles in multivalent binding events leading to macromolecular phase transitions that contribute to the formation of membrane-less organelles.
      Importantly, these varied interactions can often be readily modulated by post-translational modifications (PTMs) of the IDP/IDR (
      • Bah A.
      • Forman-Kay J.D.
      Modulation of intrinsically disordered protein function by post-translational modifications.
      ,
      • Banerjee R.
      Introduction to the thematic minireview series on intrinsically disordered proteins.
      ). Indeed, an unfolded peptide chain is typically more accessible to modifying enzymes. PTMs change the physicochemical properties of the primary sequence; this produces a variety of structural changes, which then leads to alteration and expansion of IDP function. Specifically, PTMs can alter a given protein's steric, hydrophobic, or electrostatic properties, can stabilize, destabilize, or induce local structure, and can inhibit or enhance long-range tertiary contacts. PTMs alter the energy landscape and resultant conformational ensemble of the IDP, and they modulate interactions with other biomolecules (
      • Uversky V.N.
      Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.
      ,
      • Shammas S.L.
      • Crabtree M.D.
      • Dahal L.
      • Wicky B.I.
      • Clarke J.
      Insights into coupled folding and binding mechanisms from kinetic studies.
      ,
      • Bah A.
      • Forman-Kay J.D.
      Modulation of intrinsically disordered protein function by post-translational modifications.
      ,
      • Guharoy M.
      • Bhowmick P.
      • Tompa P.
      Design principles involving protein disorder facilitate specific substrate selection and degradation by the ubiquitin-proteasome system.
      ).

      Structural disorder in vesicle trafficking

      The trafficking of synaptic vesicles is a specialized case of cellular trafficking, which in general requires that vesicles carrying the appropriate cargo bud from a source membrane, travel in the appropriate direction, and then fuse with the proper cellular target. It is therefore relevant to examine the ways in which structural disorder may contribute to general vesicle trafficking pathways before examining contexts that are more specific to trafficking at the synapse. Using primary sequence analysis, Pietrosemoli et al. (
      • Pietrosemoli N.
      • Pancsa R.
      • Tompa P.
      Structural disorder provides increased adaptability for vesicle trafficking pathways.
      ) examined protein disorder in cellular trafficking pathways such as clathrin-mediated endocytosis and transport between the ER and Golgi mediated by COPI (Golgi to ER) and COPII (ER to Golgi) coat proteins. They found that proteins associated with enzymatic activity and proteins that function as adaptors for vesicle cargo featured especially high degrees of disorder.
      Long disordered regions mediating protein/protein interactions were often found adjacent to structured catalytic domains (
      • Pietrosemoli N.
      • Pancsa R.
      • Tompa P.
      Structural disorder provides increased adaptability for vesicle trafficking pathways.
      ). Synaptojanins, for example, contain three distinct domains: an N-terminal inositol phosphatase domain; a central inositol phosphatase domain; and a disordered C-terminal domain featuring a proline-rich domain and three asparagine–proline–phenylalanine (NPF) repeats (Fig. 1A). The C-terminal domain interacts with other proteins involved in endocytosis, including the AP2 adaptor complex, amphiphysin, endophilin, DAP160/intersectin, syndaptin, and Eps15 (
      • Montesinos M.L.
      • Castellano-Muñoz M.
      • García-Junco-Clemente P.
      • Fernández-Chacón R.
      Recycling and EH domain proteins at the synapse.
      ). Importantly, synaptojanin-1 is a synapse-specific family member that plays a critical role in the endocytosis of synaptic vesicles and is also mutated in some forms of familial Parkinson's disease (
      • Drouet V.
      • Lesage S.
      Synaptojanin 1 mutation in Parkinson's disease brings further insight into the neuropathological mechanisms.
      ).
      Figure thumbnail gr1
      Figure 1Intrinsically disordered structure confers a variety of functional advantages. A, disordered C-terminal region of synaptojanin, which consists of a proline-rich domain (purple) and an asparagine–proline–phenylalanine (NPF) repeat region (orange), allows it to interact with a number of other proteins. Synaptojanin also contains two N-terminal phosphoinositide phosphatase domains (noted as Sac1p and Ins 5′ptase, in red and blue, respectively). B, ArfGAP1 contains two disordered, membrane curvature-sensing ALPS motifs (pink) that selectively bind to highly-curved membranes. Curvature-selective membrane binding by these motifs appears coupled to activation of the zinc finger GAP domain (cyan), which facilitates Arf1 GTP hydrolysis and triggers disassembly of the COPI coat. The ArfGAP1 ALPS motifs adopt helical structure upon membrane binding. C, epsin N-terminal homology domain (ENTH, brown), selectively binds to curved membranes. The disordered epsin C terminus “fly-casts” to interact with varied proteins: its disordered structure increases the range at which it can bind to and capture necessary binding targets.
      As another example, the GTPase-activating protein (GAP) ArfGAP1, which regulates the enzymatic activity of ADP-ribosylation factor 1 (Arf1), contains disordered amphipathic lipid-packing sensor (ALPS) motifs that bind to membranes and sense membrane curvature to contribute critically to COPI trafficking (
      • Ambroggio E.
      • Sorre B.
      • Bassereau P.
      • Goud B.
      • Manneville J.B.
      • Antonny B.
      ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions.
      • Antonny B.
      Mechanisms of membrane curvature sensing.
      ,
      • Bigay J.
      • Gounon P.
      • Robineau S.
      • Antonny B.
      Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature.
      ,
      • Bigay J.
      • Casella J.F.
      • Drin G.
      • Mesmin B.
      • Antonny B.
      ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif.
      ,
      • Drin G.
      • Antonny B.
      Amphipathic helices and membrane curvature.
      • Mesmin B.
      • Drin G.
      • Levi S.
      • Rawet M.
      • Cassel D.
      • Bigay J.
      • Antonny B.
      Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
      ). During COPI vesicle biogenesis, GTP-bound Arf1 binds to organelle membranes and recruits coatomer, a large cytosolic complex that gathers cargo and assembles into a vesicle coat (
      • Antonny B.
      Mechanisms of membrane curvature sensing.
      ,
      • Drin G.
      • Antonny B.
      Amphipathic helices and membrane curvature.
      ,
      • Mesmin B.
      • Drin G.
      • Levi S.
      • Rawet M.
      • Cassel D.
      • Bigay J.
      • Antonny B.
      Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
      ). Coat disassembly of mature vesicles requires GTP hydrolysis by Arf1, which is in turn stimulated by ArfGAP1 (
      • Rothman J.E.
      • Wieland F.T.
      Protein sorting by transport vesicles.
      ). The ArfGAP1 ALPS motifs bind avidly to small liposomes in an α-helical conformation, with membrane binding predominantly driven through insertion of a number of bulky hydrophobic residues on the hydrophobic face of the amphipathic helices (
      • Antonny B.
      Mechanisms of membrane curvature sensing.
      ,
      • Bigay J.
      • Casella J.F.
      • Drin G.
      • Mesmin B.
      • Antonny B.
      ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif.
      ,
      • Drin G.
      • Antonny B.
      Amphipathic helices and membrane curvature.
      • Mesmin B.
      • Drin G.
      • Levi S.
      • Rawet M.
      • Cassel D.
      • Bigay J.
      • Antonny B.
      Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
      ,
      • Drin G.
      • Casella J.F.
      • Gautier R.
      • Boehmer T.
      • Schwartz T.U.
      • Antonny B.
      A general amphipathic α-helical motif for sensing membrane curvature.
      ,
      • Vanni S.
      • Vamparys L.
      • Gautier R.
      • Drin G.
      • Etchebest C.
      • Fuchs P.F.
      • Antonny B.
      Amphipathic lipid packing sensor motifs: probing bilayer defects with hydrophobic residues.
      ). Interestingly, the GTPase-stimulating activity of ArfGAP1 increases with increasing membrane curvature (
      • Bigay J.
      • Gounon P.
      • Robineau S.
      • Antonny B.
      Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature.
      ). This suggests a feedback loop (Fig. 1B): Arf1/COPI first induce membrane deformation and so enhance membrane curvature, which in turn stimulates ArfGAP1 activity, enhances Arf1 GTP hydrolysis, and leads to coat disassembly (
      • Mesmin B.
      • Drin G.
      • Levi S.
      • Rawet M.
      • Cassel D.
      • Bigay J.
      • Antonny B.
      Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
      ). Although COPI vesicles are not present at the synapse, ArfGAP1 provides an example of how IDP/membrane interactions contribute to vesicle trafficking. Additional synapse-specific examples are discussed further below.
      An example of a disorder-containing adaptor protein is the Epsin family of proteins. Epsins contain a folded epsin N-terminal homology domain at their N terminus that interacts with membranes, whereas the rest of the protein is disordered (
      • Pietrosemoli N.
      • Pancsa R.
      • Tompa P.
      Structural disorder provides increased adaptability for vesicle trafficking pathways.
      ). At their C termini, epsins contain binding sites for clathrin, AP2 adaptors, and ubiquitinated cargos (
      • Sen A.
      • Madhivanan K.
      • Mukherjee D.
      • Aguilar R.C.
      The epsin protein family: coordinators of endocytosis and signaling.
      ). This modular architecture (Fig. 1C) allows epsin to link the membrane with other proteins involved in vesicle formation (
      • Capraro B.R.
      • Yoon Y.
      • Cho W.
      • Baumgart T.
      Curvature sensing by the epsin N-terminal homology domain measured on cylindrical lipid membrane tethers.
      ) and to thereby aid in cargo selection. Epsins may employ a fly-casting type mechanism (
      • Pietrosemoli N.
      • Pancsa R.
      • Tompa P.
      Structural disorder provides increased adaptability for vesicle trafficking pathways.
      ), with a protein-binding site at one end of a disordered chain. The larger resulting hydrodynamic radius allows a binding module to explore a greater volume and thereby helps increase association rates (
      • Cortese M.S.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in scaffold proteins: getting more from less.
      ,
      • Shoemaker B.A.
      • Portman J.J.
      • Wolynes P.G.
      Speeding molecular recognition by using the folding funnel: the fly-casting mechanism.
      ). Like synaptojanin-1, epsin-1 is thought to play a role in clathrin-mediated synaptic vesicle endocytosis/recycling.

      Intrinsically disordered SNARE proteins as the core membrane fusion machinery

      The SNARE proteins (for SNAP receptor proteins, where SNAPs are soluble NSF attachment proteins, and NSF is the N-ethylmaleimide-sensitive factor) are a superfamily of small proteins that mediate membrane fusion in all steps of cellular secretory pathways and that constitute the core membrane fusion machinery (
      • Jahn R.
      • Scheller R.H.
      SNAREs–engines for membrane fusion.
      ,
      • Südhof T.C.
      • Rizo J.
      Synaptic vesicle exocytosis.
      ). Humans contain 36 different SNARE proteins, which are found attached to membranes, often through a C-terminal transmembrane domain, although attachment via post-translational lipidation also occurs for some family members. SNAREs are often classified as v- or t-SNAREs depending on their localization to vesicles or to the target membrane, respectively. Membrane fusion events obviously are critical for synaptic vesicle exocytosis, where the most relevant SNAREs are the v-SNARE synaptobrevin-2 and the t-SNAREs syntaxin-1 and synaptosome-associated protein of 25 kDa (SNAP-25).
      The SNARE motif is a conserved stretch of 60–70 amino acids featuring heptad repeats (
      • Jahn R.
      • Scheller R.H.
      SNAREs–engines for membrane fusion.
      ). Notably, free SNARE motifs are disordered in their free state both in vitro and in live cells (
      • Fasshauer D.
      • Otto H.
      • Eliason W.K.
      • Jahn R.
      • Brünger A.T.
      Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation.
      • Fasshauer D.
      • Bruns D.
      • Shen B.
      • Jahn R.
      • Brünger A.T.
      A structural change occurs upon binding of syntaxin to SNAP-25.
      ,
      • Fasshauer D.
      Structural insights into the SNARE mechanism.
      ,
      • Hazzard J.
      • Südhof T.C.
      • Rizo J.
      NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin.
      ,
      • Margittai M.
      • Fasshauer D.
      • Pabst S.
      • Jahn R.
      • Langen R.
      Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling.
      ,
      • Sakon J.J.
      • Weninger K.R.
      Detecting the conformation of individual proteins in live cells.
      • Ungar D.
      • Hughson F.M.
      SNARE protein structure and function.
      ). SNARE motifs localized to two closely apposed membranes can zipper in an N- to C-terminal direction into a parallel four-helix coiled coil termed the SNARE complex. The coiled-coil interface consists of 16 stacked layers of interacting side chains, mostly featuring hydrophobic interactions, with a central “0” layer that contains three highly-conserved glutamine residues and one highly-conserved arginine residue (SNAREs may also be classified as Qa-, Qb-, Qc-, and R-SNAREs depending on the identity of this residue) (
      • Jahn R.
      • Scheller R.H.
      SNAREs–engines for membrane fusion.
      ). The free energy released through the SNARE assembly process is thought to drive fusion of the two membranes, although precisely how the energetics of SNARE assembly are coupled to membrane fusion remains unclear. In one model, SNARE complex folding may subsequently propagate through the transmembrane domain to exert a mechanical force on fusing membranes (illustrated using the synaptic SNARE proteins in Fig. 2); this has yet to be definitively established, however (
      • Rizo J.
      • Südhof T.C.
      The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices–guilty as charged?.
      ,
      • Jahn R.
      • Fasshauer D.
      Molecular machines governing exocytosis of synaptic vesicles.
      ,
      • Südhof T.C.
      Neurotransmitter release: the last millisecond in the life of a synaptic vesicle.
      ). Regardless, SNAREs represent a clear example of how coupled binding and folding by a disordered region fulfills a critical cellular function. After assembly, the AAA ATPase NSF, along with SNAP proteins, disassembles post-fusion SNARE complexes to “recharge” the system with the necessary energy required for future rounds of fusion (
      • Südhof T.C.
      • Rizo J.
      Synaptic vesicle exocytosis.
      ).
      Figure thumbnail gr2
      Figure 2SNARE proteins constitute a core membrane fusion machinery wherein a disorder-to-order transition is coupled to force transfer that induces membrane fusion. The SNARE proteins associated with synaptic vesicle exocytosis are highlighted here. Initially, syntaxin-1 (red) and SNAP-25 (gray) are anchored to the plasma membrane, whereas synaptobrevin-2 (blue) is anchored to synaptic vesicles. During synaptic vesicle fusion with the plasma membrane, the disordered SNARE motifs of these three proteins assemble into a stable four-helix bundle (with two helices contributed by SNAP-25). Although still incompletely understood, the energy of SNARE complex assembly is thought to drive membrane fusion, as the complex transitions from an initial partially assembled trans-SNARE complex (with SNAREs anchored in opposing membranes), through fusion pore opening, to a final fully assembled cis-SNARE complex (with all three SNAREs now in the plasma membrane). Subsequent to membrane fusion, the cis-SNARE complex is disassembled by NSF in an ATP-dependent fashion.
      SNARE assembly is likely an ordered, sequential reaction involving delivery of the appropriate SNARE in the right condition, at the right place, and time (
      • Jahn R.
      • Scheller R.H.
      SNAREs–engines for membrane fusion.
      ). In addition, as noted above, SNARE assembly occurs through N- to C-terminal zippering, and this process can be frozen at intermediate stages by protein-binding partners (e.g. complexin, discussed below). Such intermediate species may include partially zippered SNARE complexes, binary and/or ternary SNARE complexes, or complexes containing SNARE protein(s) along with one or more non-SNARE–binding partners (
      • Rizo J.
      • Südhof T.C.
      The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices–guilty as charged?.
      ,
      • Rizo J.
      • Xu J.
      The synaptic vesicle release machinery.
      ,
      • Jahn R.
      • Scheller R.H.
      SNAREs–engines for membrane fusion.
      ,
      • Südhof T.C.
      Neurotransmitter release: the last millisecond in the life of a synaptic vesicle.
      ,
      • Ma C.
      • Su L.
      • Seven A.B.
      • Xu Y.
      • Rizo J.
      Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release.
      ). Indeed, for proper assembly, SNAREs must interact with a number of other proteins, and in many cases these interactions involve some degree of SNARE folding. Thus, their structural flexibility likely allows the SNARE proteins to engage a variety of partners through discrete bound state conformations and in various multimeric states, a feat that is critical for their proper regulation and function.

      Structural disorder within accessory proteins that regulate synaptic vesicle exocytosis

      The three synaptic SNARE proteins alone are sufficient to drive some degree of membrane fusion in in vitro fusion assays. In vivo, however, their behavior is tightly regulated and modulated by a number of accessory proteins, including complexin, synaptotagmin (the calcium sensor that triggers action potential evoked exocytosis by coupling calcium influx to SNARE assembly), Munc18, Munc13, tomosyn, RIM proteins, RIM-binding proteins (RIM-BPs), Rab proteins, ELKS, α-Liprins, and Piccolo/Bassoon proteins (
      • Rizo J.
      • Rosenmund C.
      Synaptic vesicle fusion.
      ,
      • Rizo J.
      • Südhof T.C.
      The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices–guilty as charged?.
      ,
      • Südhof T.C.
      The presynaptic active zone.
      ,
      • Südhof T.C.
      Neurotransmitter release: the last millisecond in the life of a synaptic vesicle.
      ,
      • Brose N.
      For better or for worse: complexins regulate SNARE function and vesicle fusion.
      ,
      • Hatsuzawa K.
      • Lang T.
      • Fasshauer D.
      • Bruns D.
      • Jahn R.
      The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis.
      • Bielopolski N.
      • Lam A.D.
      • Bar-On D.
      • Sauer M.
      • Stuenkel E.L.
      • Ashery U.
      Differential interaction of tomosyn with syntaxin and SNAP25 depends on domains in the WD40 β-propeller core and determines its inhibitory activity.
      ). This extensive machinery utilizes abundant protein/protein and protein/membrane interactions to structurally and functionally organize active zones, specialized electron-dense presynaptic membrane-associated sites of synaptic vesicle exocytosis that appose post-synaptic densities (the reader is referred to Refs.
      • Südhof T.C.
      The presynaptic active zone.
      ,
      • Ungar D.
      • Hughson F.M.
      SNARE protein structure and function.
      for further information regarding active-zone proteins, composition, organization, and architecture). At active zones, synaptic vesicles are docked and primed adjacent to the requisite calcium channels so that evoked fusion can occur efficiently when required, with a tight and rapid (sub-millisecond) coupling between the influx of calcium that results from the arrival of an action potential and vesicle release (
      • Südhof T.C.
      The presynaptic active zone.
      ,
      • Südhof T.C.
      Neurotransmitter release: the last millisecond in the life of a synaptic vesicle.
      ). SNAREs can also mediate low levels of spontaneous synaptic vesicle fusion with the plasma membrane, but for optimal action-potential–based inter-neuronal signaling, such spontaneous exocytosis must be kept to a minimum by regulatory proteins (
      • Martin J.A.
      • Hu Z.
      • Fenz K.M.
      • Fernandez J.
      • Dittman J.S.
      Complexin has opposite effects on two modes of synaptic vesicle fusion.
      ). Importantly, intrinsically disordered regions from a number of SNARE accessory/regulatory proteins play key functional roles within this extensive interaction network to help organize the active zone and optimize the synapse for rapid exocytosis.

      Tomosyn

      Tomosyn is a SNARE-binding presynaptic protein that is considered to be an inhibitor of synaptic vesicle release, possibly at the priming step. Tomosyn contains two conserved domains, including a C-terminal region with homology to the R-SNAREs, and an N-terminal WD40 repeat region (Fig. 3A). As with other SNAREs, the tomosyn SNARE motif is disordered in the free state, but it can dimerize with syntaxin-1 (
      • Fujita Y.
      • Shirataki H.
      • Sakisaka T.
      • Asakura T.
      • Ohya T.
      • Kotani H.
      • Yokoyama S.
      • Nishioka H.
      • Matsuura Y.
      • Mizoguchi A.
      • Scheller R.H.
      • Takai Y.
      Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.
      ) or can compete with synaptobrevin-2 to form a ternary SNARE complex with syntaxin-1 and SNAP-25 (
      • Bielopolski N.
      • Lam A.D.
      • Bar-On D.
      • Sauer M.
      • Stuenkel E.L.
      • Ashery U.
      Differential interaction of tomosyn with syntaxin and SNAP25 depends on domains in the WD40 β-propeller core and determines its inhibitory activity.
      ). Interestingly, two unstructured loops in the WD40 domain contribute to SNAP-25 binding and ternary SNARE complex formation by tomosyn, but not to tomosyn/syntaxin-1 dimer formation (
      • Bielopolski N.
      • Lam A.D.
      • Bar-On D.
      • Sauer M.
      • Stuenkel E.L.
      • Ashery U.
      Differential interaction of tomosyn with syntaxin and SNAP25 depends on domains in the WD40 β-propeller core and determines its inhibitory activity.
      ). Through ternary SNARE complex formation, tomosyn can regulate synaptobrevin-2 binding to syntaxin-1 and SNAP-25, and this R-SNARE substitution mechanism may underlie the inhibition of exocytosis by tomosyn overexpression in PC12 cells (
      • Hatsuzawa K.
      • Lang T.
      • Fasshauer D.
      • Bruns D.
      • Jahn R.
      The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis.
      ). Alternatively, tomosyn binding to the SNARE motif of syntaxin-1 may displace Munc18 from syntaxin; it was proposed that tomosyn may activate syntaxin-1 and allow it to interact with synaptobrevin-2 (
      • Fujita Y.
      • Shirataki H.
      • Sakisaka T.
      • Asakura T.
      • Ohya T.
      • Kotani H.
      • Yokoyama S.
      • Nishioka H.
      • Matsuura Y.
      • Mizoguchi A.
      • Scheller R.H.
      • Takai Y.
      Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.
      ).
      Figure thumbnail gr3
      Figure 3Functional mechanisms of disorder in synaptic proteins. A, tomosyn contains a disordered C-terminal R-SNARE domain (blue). Its N-terminal WD40 repeat domain (orange), which features β-propeller structures, contains unstructured loops necessary for a subset of tomosyn functions. B, segregation of positively and negatively charged residues within an unstructured region of synaptotagmin allows it to behave as a molecular zipper whose structural state modulates synaptotagmin function: in the open state, synaptotagmin contributes to vesicle docking, and in the zippered state, it facilitates membrane fusion. C, Munc18 contains an unstructured loop (pink) within domain 3a that allows helix 12 of this domain to shorten or extend and thereby toggle between syntaxin binding and inhibition of membrane fusion or synaptobrevin-2 binding and promotion of fusion, respectively. D, some mammalian homologs of synapse–defective-1 proteins contain a disordered domain (brown) that interacts with other proteins, including Munc18-1 and the LAR–Liprin A2 complex. They also contain a central C2 domain (yellow) and a C-terminal RhoGAP domain (green).

      Synaptotagmin

      Synaptotagmin-1 (Syt1) has been identified as a key calcium sensor linking a presynaptic membrane depolarization-induced calcium influx to evoked exocytosis. Syt1 contains an N-terminal transmembrane α-helix followed by two C2 domains, C2A and C2B, which bind three and two Ca2+ ions, respectively, through calcium-binding loops that can then interact with membranes. Syt1 thus interacts with phospholipid membranes in a Ca2+-dependent manner. Syt1 also interacts with SNAREs and can bind simultaneously to membranes and membrane-anchored SNARE complexes to form the so-called quaternary SNARE–synaptotagmin-1–Ca2+–phospholipid (SSCAP) complex (
      • Rizo J.
      • Rosenmund C.
      Synaptic vesicle fusion.
      ). It is unclear, however, whether the synaptotagmin/SNARE interaction is mediated by a polybasic region on the side of the C2B domain (
      • Rizo J.
      • Rosenmund C.
      Synaptic vesicle fusion.
      ,
      • Brewer K.D.
      • Bacaj T.
      • Cavalli A.
      • Camilloni C.
      • Swarbrick J.D.
      • Liu J.
      • Zhou A.
      • Zhou P.
      • Barlow N.
      • Xu J.
      • Seven A.B.
      • Prinslow E.A.
      • Voleti R.
      • Häussinger D.
      • Bonvin A.M.
      • et al.
      Dynamic binding mode of a synaptotagmin-1–SNARE complex in solution.
      ) or by a region of C2B opposite the calcium-binding loops containing two critical arginine residues (
      • Brewer K.D.
      • Bacaj T.
      • Cavalli A.
      • Camilloni C.
      • Swarbrick J.D.
      • Liu J.
      • Zhou A.
      • Zhou P.
      • Barlow N.
      • Xu J.
      • Seven A.B.
      • Prinslow E.A.
      • Voleti R.
      • Häussinger D.
      • Bonvin A.M.
      • et al.
      Dynamic binding mode of a synaptotagmin-1–SNARE complex in solution.
      ,
      • Zhou Q.
      • Lai Y.
      • Bacaj T.
      • Zhao M.
      • Lyubimov A.Y.
      • Uervirojnangkoorn M.
      • Zeldin O.B.
      • Brewster A.S.
      • Sauter N.K.
      • Cohen A.E.
      • Soltis S.M.
      • Alonso-Mori R.
      • Chollet M.
      • Lemke H.T.
      • Pfuetzner R.A.
      • et al.
      Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis.
      • Zhou Q.
      • Zhou P.
      • Wang A.L.
      • Wu D.
      • Zhao M.
      • Südhof T.C.
      • Brunger A.T.
      The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis.
      ). This complex could facilitate fusion by inducing negative membrane curvature, with the Syt1/membrane interaction serving to bring apposed membranes together. Alternatively, Syt1 may induce positive curvature in the plasma membrane. Interestingly, and similarly to complexin, Syt1 (and Syt2) also normally clamp spontaneous SV release, and the activating versus inhibitory functions of synaptotagmin are likely independent (
      • Südhof T.C.
      Neurotransmitter release: the last millisecond in the life of a synaptic vesicle.
      ).
      An intrinsically disordered region between the synaptotagmin transmembrane domain and C2A has been shown to be essential for both calcium-independent vesicle docking, and calcium-dependent fusion pore opening (
      • Lai Y.
      • Lou X.
      • Jho Y.
      • Yoon T.Y.
      • Shin Y.K.
      The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening.
      ). This region interacts with lipid membranes, and it modulates calcium binding within C2A, consistent with the idea that IDRs often influence adjacent folded domains (
      • Fealey M.E.
      • Mahling R.
      • Rice A.M.
      • Dunleavy K.
      • Kobany S.E.
      • Lohese K.J.
      • Horn B.
      • Hinderliter A.
      Synaptotagmin I's intrinsically disordered region interacts with synaptic vesicle lipids and exerts allosteric control over C2A.
      ). Interestingly, this IDR contains an N-terminal part rich in basic amino acids and a C-terminal part rich in acidic amino acids. This segregation of charge seems to result in a molecular zipper that contributes to fusion pore opening when closed but that facilitates vesicle docking when open (Fig. 3B); shortening the linker or cross-linking it into a folded conformation reduces docking but enhances fusion pore opening. These observations led to a model whereby this disordered linker region extends to facilitate vesicle docking (perhaps through a sort of fly-casting mechanism) but then folds in a way that facilitates fusion pore opening (
      • Lai Y.
      • Lou X.
      • Jho Y.
      • Yoon T.Y.
      • Shin Y.K.
      The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening.
      ).
      Finally, although C2A is a folded domain of synaptotagmin (
      • Gauer J.W.
      • Sisk R.
      • Murphy J.R.
      • Jacobson H.
      • Sutton R.B.
      • Gillispie G.D.
      • Hinderliter A.
      Mechanism for calcium ion sensing by the C2A domain of synaptotagmin I.
      ), it appears to be weakly stable. It was proposed that this marginal stability allows C2A to adopt and fluctuate between multiple conformational states with unique functions and behaviors, which endow synaptotagmin with great functional diversity (
      • Gauer J.W.
      • Sisk R.
      • Murphy J.R.
      • Jacobson H.
      • Sutton R.B.
      • Gillispie G.D.
      • Hinderliter A.
      Mechanism for calcium ion sensing by the C2A domain of synaptotagmin I.
      ).

      Munc18

      SM proteins (for Sec1/Munc18-like proteins) are critical partners in all SNARE-mediated membrane fusion events, although how they contribute to membrane fusion remains enigmatic (
      • Rizo J.
      • Rosenmund C.
      Synaptic vesicle fusion.
      ,
      • Rizo J.
      • Südhof T.C.
      The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices–guilty as charged?.
      ). At least three primary functions have been proposed for Munc18-1 (
      • Han G.A.
      • Bin N.R.
      • Kang S.Y.
      • Han L.
      • Sugita S.
      Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.
      ). 1) It acts as a chaperone for syntaxin-1 that facilitates proper syntaxin-1 localization and expression. Indeed, in contrast to its apparently essential facilitatory role in synaptic vesicle fusion, Munc18 was originally shown to bind to the “closed” conformation of syntaxin in a way that would be expected to inhibit membrane fusion (with the N-terminal Habc domain of syntaxin folded back onto the SNARE domain). In this conformation, Munc18 stabilizes syntaxin-1 and facilitates transport of syntaxin-1 to the plasma membrane. 2) Munc18 facilitates priming via promotion of SNARE-mediated membrane fusion in several different ways. Munc18 was later shown to bind to the syntaxin N-peptide (at the very N terminus) in a conformation that is compatible with SNARE complex assembly. It also binds syntaxin–1-SNAP-25 heterodimers and could thus facilitate or nucleate SNARE complex assembly. In addition to binding t-SNAREs like syntaxin, it can also simultaneously bind to v-SNAREs, possibly aligning them so as to facilitate the initial steps of SNARE complex formation (
      • Baker R.W.
      • Jeffrey P.D.
      • Zick M.
      • Phillips B.P.
      • Wickner W.T.
      • Hughson F.M.
      A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly.
      ,
      • Parisotto D.
      • Pfau M.
      • Scheutzow A.
      • Wild K.
      • Mayer M.P.
      • Malsam J.
      • Sinning I.
      • Sollner T.H.
      An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly.
      ). Furthermore it may enable lipid mixing either directly or through lipid destabilization, and it may help to spatially and asymmetrically organize assembled SNARE complexes around the fusion site by preventing diffusion of the bulky Munc18–SNARE complex assembly to the center of the synaptic vesicle/plasma membrane intermembrane space. 3) Finally, it has been suggested that Munc18 contributes to docking of large dense-core vesicles (
      • Rizo J.
      • Rosenmund C.
      Synaptic vesicle fusion.
      ,
      • Rizo J.
      • Südhof T.C.
      The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices–guilty as charged?.
      ,
      • Südhof T.C.
      • Rizo J.
      Synaptic vesicle exocytosis.
      ,
      • Han G.A.
      • Bin N.R.
      • Kang S.Y.
      • Han L.
      • Sugita S.
      Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.
      ).
      The Munc18 sequence can be subdivided into domains 1, 2, 3a, and 3b (Fig. 3C) (
      • Martin S.
      • Tomatis V.M.
      • Papadopulos A.
      • Christie M.P.
      • Malintan N.T.
      • Gormal R.S.
      • Sugita S.
      • Martin J.L.
      • Collins B.M.
      • Meunier F.A.
      The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane.
      ). In the Munc18/syntaxin-1 binary structure, closed syntaxin interacts with a Munc18 cavity formed by domains 1 and 3 (
      • Parisotto D.
      • Pfau M.
      • Scheutzow A.
      • Wild K.
      • Mayer M.P.
      • Malsam J.
      • Sinning I.
      • Sollner T.H.
      An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly.
      ). Domain 1 plays an important role in the syntaxin chaperoning function of Munc18 and so-called “chaperoning mutants” can be generated within this domain (
      • Han G.A.
      • Bin N.R.
      • Kang S.Y.
      • Han L.
      • Sugita S.
      Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.
      ). In contrast, domain 3a appears to play a role in the priming function of Munc18, and mutations in this domain can impair priming (“priming mutants”) (
      • Han G.A.
      • Bin N.R.
      • Kang S.Y.
      • Han L.
      • Sugita S.
      Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.
      ). Interestingly, in the Munc18–syntaxin-1 complex, two anti-parallel helices in domain 3a, helices 11 and 12, are connected by a 21-residue bent hairpin loop with an irregular conformation. Eight residues of this loop are disordered as they do not appear in crystal structures, and the remaining visible residues have high B-factors, indicating increased mobility. This domain 3a loop is essential for exocytosis from PC12 cells but is not required for syntaxin-1A transport (
      • Martin S.
      • Tomatis V.M.
      • Papadopulos A.
      • Christie M.P.
      • Malintan N.T.
      • Gormal R.S.
      • Sugita S.
      • Martin J.L.
      • Collins B.M.
      • Meunier F.A.
      The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane.
      ). In structures of Munc18 bound to closed syntaxin, helix 12 is relatively short (
      • Colbert K.N.
      • Hattendorf D.A.
      • Weiss T.M.
      • Burkhardt P.
      • Fasshauer D.
      • Weis W.I.
      Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation.
      ), but in structures bound to the syntaxin N peptide helix 12 forms a longer, extended helix (
      • Hu S.H.
      • Christie M.P.
      • Saez N.J.
      • Latham C.F.
      • Jarrott R.
      • Lua L.H.
      • Collins B.M.
      • Martin J.L.
      Possible roles for Munc18-1 domain 3a and syntaxin1 N-peptide and C-terminal anchor in SNARE complex formation.
      ). Mutations that bias extension of helix 12 increase binding to synaptobrevin-2 and enhance priming by Munc18, suggesting that the extended helix 12 provides a template for SNARE assembly (
      • Baker R.W.
      • Jeffrey P.D.
      • Zick M.
      • Phillips B.P.
      • Wickner W.T.
      • Hughson F.M.
      A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly.
      ,
      • Parisotto D.
      • Pfau M.
      • Scheutzow A.
      • Wild K.
      • Mayer M.P.
      • Malsam J.
      • Sinning I.
      • Sollner T.H.
      An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly.
      ). The disordered residues in the loop connecting helices 11 and 12 thus appear to contribute to the structural flexibility necessary for Munc18 to pivot between the requisite structures for its inhibitory and facilitatory functions (Fig. 3C).

      Mouse synapse-defective 1A

      Synapse–defective-1 proteins, including mSYD1A (mouse synapse–defective-1A), regulate presynaptic differentiation. Many SYD-1 proteins contain a RhoGAP domain and a PDZ domain that links SYD-1 to the surface receptor neurexin (a presynaptic protein that helps connect neurons across the synaptic cleft); these domains may mediate SYD-1 function. Syd1 mutations in Caenorhabditis elegans perturb proper localization of active-zone components and synaptic vesicles (
      • Wentzel C.
      • Sommer J.E.
      • Nair R.
      • Stiefvater A.
      • Sibarita J.B.
      • Scheiffele P.
      mSYD1A, a mammalian synapse-defective-1 protein, regulates synaptogenic signaling and vesicle docking.
      ). The first mammalian SYD-1 ortholog was recently identified in mice (mSYD1A) and shown to contribute to vesicle docking, insofar as mSYD1A knockout hippocampal synapses contain fewer docked vesicles and reduced synaptic transmission. Interestingly, mSYD1 function depends on a multifunctional intrinsically disordered domain (Fig. 3D). In contrast to invertebrate SYD-1, mSYD1A lacks the PDZ domain and contains an active RhoGAP domain (which is inactive in invertebrates). mSYD1A RhoGAP activity is regulated through intramolecular interactions that include the disordered domain. Furthermore, the disordered domain acts as an interaction module capable of interacting with multiple proteins, including Munc18-1 as well as a LAR–LiprinA2 complex. The protein/protein interactions mediated by this disordered domain likely contribute to organization of the active zone and to proper vesicle docking (
      • Wentzel C.
      • Sommer J.E.
      • Nair R.
      • Stiefvater A.
      • Sibarita J.B.
      • Scheiffele P.
      mSYD1A, a mammalian synapse-defective-1 protein, regulates synaptogenic signaling and vesicle docking.
      ).
      The examples above illustrate both the importance of disordered protein regions in the regulation of SNARE-mediated synaptic vesicle exocytosis and some of the mechanisms involved. Disordered regions can compete with other disordered binding motifs for binding partners, can served as a platform for recruiting multiple interaction partners to provide scaffolding/organizing functions, and can undergo highly-specific or less well-defined conformational transitions that regulate function and activity. The diversity of their modes of action contributes to the ubiquitous involvement of disordered proteins in various biological processes, but also constitutes a challenge in uncovering and understanding their functional roles.

      Protein/lipid interactions at the synapse: functional significance of membrane binding by IDPs

      A number of IDPs interact with lipid membranes. This interaction can be driven by partitioning of hydrophobic residues into the membrane as well as by electrostatic interactions between charged protein residues and lipid headgroups (
      • Seelig J.
      Thermodynamics of lipid-peptide interactions.
      ,
      • White S.H.
      • Wimley W.C.
      Hydrophobic interactions of peptides with membrane interfaces.
      • White S.H.
      • Wimley W.C.
      Membrane protein folding and stability: physical principles.
      ). As with IDP/protein interactions, in many cases the IDP folds upon membrane binding, often into an amphipathic membrane-binding helix (
      • Drin G.
      • Casella J.F.
      • Gautier R.
      • Boehmer T.
      • Schwartz T.U.
      • Antonny B.
      A general amphipathic α-helical motif for sensing membrane curvature.
      ). Some IDPs instead remain unfolded in the membrane-bound state, as is the case, for example, with the MARCKS peptide (a membrane-binding region of the myristoylated alanine-rich protein kinase C substrate, or MARCKS, protein, which can sequester phosphatidylinositol 4,5-bisphosphate (PIP2) in the membrane and regulate phospholipase C signaling) (
      • Morton L.A.
      • Tamura R.
      • de Jesus A.J.
      • Espinoza A.
      • Yin H.
      Biophysical investigations with MARCKS-ED: dissecting the molecular mechanism of its curvature sensing behaviors.
      ). The functional roles of IDP/membrane binding are not always clear; in some cases, it may contribute to proper cellular localization (
      • Ambroggio E.
      • Sorre B.
      • Bassereau P.
      • Goud B.
      • Manneville J.B.
      • Antonny B.
      ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions.
      ,
      • Bigay J.
      • Casella J.F.
      • Drin G.
      • Mesmin B.
      • Antonny B.
      ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif.
      ,
      • Mesmin B.
      • Drin G.
      • Levi S.
      • Rawet M.
      • Cassel D.
      • Bigay J.
      • Antonny B.
      Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
      ,
      • Wragg R.T.
      • Snead D.
      • Dong Y.
      • Ramlall T.F.
      • Menon I.
      • Bai J.
      • Eliezer D.
      • Dittman J.S.
      Synaptic vesicles position complexin to block spontaneous fusion.
      ), and in others the IDP/membrane interaction may actively remodel the membrane in functionally significant ways (
      • McMahon H.T.
      • Gallop J.L.
      Membrane curvature and mechanisms of dynamic cell membrane remodelling.
      ). A number of intrinsically disordered membrane-binding proteins function as membrane curvature sensors and membrane curvature generators. Above, ArfGAP1 was cited as an example of how membrane curvature-selective binding contributes to IDP function in the context of vesicle trafficking. This type of behavior is observed at the synapse as well, as will be discussed for two intrinsically disordered pre-synaptic membrane curvature sensors, α-synuclein and complexin.

      Complexin

      Complexins form a family of small, highly-charged, and highly-conserved cytoplasmic proteins that have emerged as key regulators of SNARE-mediated synaptic vesicle exocytosis (
      • Brose N.
      For better or for worse: complexins regulate SNARE function and vesicle fusion.
      ,
      • Takahashi S.
      • Yamamoto H.
      • Matsuda Z.
      • Ogawa M.
      • Yagyu K.
      • Taniguchi T.
      • Miyata T.
      • Kaba H.
      • Higuchi T.
      • Okutani F.
      Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses.
      • McMahon H.T.
      • Missler M.
      • Li C.
      • Südhof T.C.
      Complexins: cytosolic proteins that regulate SNAP receptor function.
      ,
      • Ishizuka T.
      • Saisu H.
      • Odani S.
      • Abe T.
      Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals.
      ,
      • Ishizuka T.
      • Saisu H.
      • Suzuki T.
      • Kirino Y.
      • Abe T.
      Molecular cloning of synaphins/complexins, cytosolic proteins involved in transmitter release, in the electric organ of an electric ray (Narke japonica).
      • Reim K.
      • Wegmeyer H.
      • Brandstätter J.H.
      • Xue M.
      • Rosenmund C.
      • Dresbach T.
      • Hofmann K.
      • Brose N.
      Structurally and functionally unique complexins at retinal ribbon synapses.
      ). Although complexins are intrinsically disordered in their free state, they can directly interact with assembled and/or assembling SNARE complexes through a central α-helical domain that binds in an antiparallel fashion in the groove formed between syntaxin-1 and synaptobrevin-2 (
      • Pabst S.
      • Hazzard J.W.
      • Antonin W.
      • Südhof T.C.
      • Jahn R.
      • Rizo J.
      • Fasshauer D.
      Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions.
      ,
      • Chen X.
      • Tomchick D.R.
      • Kovrigin E.
      • Araç D.
      • Machius M.
      • Südhof T.C.
      • Rizo J.
      Three-dimensional structure of the complexin/SNARE complex.
      • Bracher A.
      • Kadlec J.
      • Betz H.
      • Weissenhorn W.
      X-ray structure of a neuronal complexin–SNARE complex from squid.
      ). Complexins feature four different domains with discrete functions in synaptic vesicle exocytosis, including a facilitatory N-terminal domain (NTD), an inhibitory accessory helix/accessory domain (AH), the above-mentioned essential central helix (CH), and a C-terminal domain (CTD) that can bind lipid membranes and that may inhibit or facilitate exocytosis (
      • Brose N.
      For better or for worse: complexins regulate SNARE function and vesicle fusion.
      ). Notably, although the NTD and CTD are highly disordered, the CH and AH domains feature a substantial population of helical structure, even in the absence of any intermolecular interaction (
      • Pabst S.
      • Hazzard J.W.
      • Antonin W.
      • Südhof T.C.
      • Jahn R.
      • Rizo J.
      • Fasshauer D.
      Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions.
      ,
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ). Such residual secondary structure elements, sometimes denoted as SLiMs (short linear motifs), MoRFs (molecular recognition features), or PreSMos (prestructured motifs) (
      • Mohan A.
      • Oldfield C.J.
      • Radivojac P.
      • Vacic V.
      • Cortese M.S.
      • Dunker A.K.
      • Uversky V.N.
      Analysis of molecular recognition features (MoRFs).
      ,
      • Hunt T.
      Sequence motifs involved in recognition and targeting: a new series.
      • Lee S.H.
      • Kim D.H.
      • Han J.J.
      • Cha E.J.
      • Lim J.E.
      • Cho Y.J.
      • Lee C.
      • Han K.H.
      Understanding pre-structured motifs (PreSMos) in intrinsically unfolded proteins.
      ), often promote or facilitate the binding of IDPs/IDRs to their interaction partners.
      Complexin expression levels are altered in a variety of neurological and psychiatric disorders. Although such expression level changes may not be causal in these disorders, they could contribute to their corresponding symptomatology (
      • Brose N.
      Altered complexin expression in psychiatric and neurological disorders: cause or consequence?.
      ). Recently, homozygous mutations in the complexin CTD have been identified through whole exome sequencing in patients with severe infantile myoclonic epilepsy and intellectual disability (
      • Redler S.
      • Strom T.M.
      • Wieland T.
      • Cremer K.
      • Engels H.
      • Distelmaier F.
      • Schaper J.
      • Küchler A.
      • Lemke J.R.
      • Jeschke S.
      • Schreyer N.
      • Sticht H.
      • Koch M.
      • Lüdecke H.J.
      • Wieczorek D.
      Variants in CPLX1 in two families with autosomal-recessive severe infantile myoclonic epilepsy and ID.
      ). Complexin-1 and -2 double knockout mice die shortly after birth, likely from deficits in synaptic transmission in multiple neuronal networks (
      • Brose N.
      For better or for worse: complexins regulate SNARE function and vesicle fusion.
      ,
      • Reim K.
      • Mansour M.
      • Varoqueaux F.
      • McMahon H.T.
      • Südhof T.C.
      • Brose N.
      • Rosenmund C.
      Complexins regulate a late step in Ca2+-dependent neurotransmitter release.
      ). Single knockout of complexin-1 or -2 is not lethal but causes neurological impairments (
      • Reim K.
      • Mansour M.
      • Varoqueaux F.
      • McMahon H.T.
      • Südhof T.C.
      • Brose N.
      • Rosenmund C.
      Complexins regulate a late step in Ca2+-dependent neurotransmitter release.
      • Glynn D.
      • Drew C.J.
      • Reim K.
      • Brose N.
      • Morton A.J.
      Profound ataxia in complexin I knockout mice masks a complex phenotype that includes exploratory and habituation deficits.
      ,
      • Glynn D.
      • Reim K.
      • Brose N.
      • Morton A.J.
      Depletion of complexin II does not affect disease progression in a mouse model of Huntington's disease (HD); support for role for complexin II in behavioural pathology in a mouse model of HD.
      ,
      • Drew C.J.
      • Kyd R.J.
      • Morton A.J.
      Complexin 1 knockout mice exhibit marked deficits in social behaviours but appear to be cognitively normal.
      • Zhao L.
      • Burkin H.R.
      • Shi X.
      • Li L.
      • Reim K.
      • Miller D.J.
      Complexin I is required for mammalian sperm acrosomal exocytosis.
      ). Complexin appears to either promote or inhibit vesicle fusion depending on the experimental approach used; complexin also appears to differentially affect action potential-evoked versus spontaneous vesicle exocytosis and to do so in a species-dependent fashion. Overall, a general consensus has emerged that complexins inhibit spontaneous release while facilitating synchronous, evoked release (
      • Brose N.
      For better or for worse: complexins regulate SNARE function and vesicle fusion.
      ,
      • Reim K.
      • Mansour M.
      • Varoqueaux F.
      • McMahon H.T.
      • Südhof T.C.
      • Brose N.
      • Rosenmund C.
      Complexins regulate a late step in Ca2+-dependent neurotransmitter release.
      ). The detailed mechanisms by which complexin fulfills these distinct functions, however, remain unclear. Regardless, complexin's inhibitory and facilitatory functions appear to be distinct and separable and so likely operate through discrete mechanisms (
      • Buhl L.K.
      • Jorquera R.A.
      • Akbergenova Y.
      • Huntwork-Rodriguez S.
      • Volfson D.
      • Littleton J.T.
      Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of complexin.
      ,
      • Cho R.W.
      • Kümmel D.
      • Li F.
      • Baguley S.W.
      • Coleman J.
      • Rothman J.E.
      • Littleton J.T.
      Genetic analysis of the complexin trans-clamping model for cross-linking SNARE complexes in vivo.
      • Lai Y.
      • Diao J.
      • Cipriano D.J.
      • Zhang Y.
      • Pfuetzner R.A.
      • Padolina M.S.
      • Brunger A.T.
      Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms.
      ). As noted above, the four complexin domains likely have discrete functions in vesicle exocytosis, and complexin domain function has been extensively dissected. Here, we focus on the highly-disordered NTD and CTD, while noting the importance of the structured CH and AH domains.
      The complexin NTD has been reported to facilitate vesicle release (
      • Lai Y.
      • Diao J.
      • Cipriano D.J.
      • Zhang Y.
      • Pfuetzner R.A.
      • Padolina M.S.
      • Brunger A.T.
      Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms.
      ,
      • Xue M.
      • Reim K.
      • Chen X.
      • Chao H.T.
      • Deng H.
      • Rizo J.
      • Brose N.
      • Rosenmund C.
      Distinct domains of complexin I differentially regulate neurotransmitter release.
      • Maximov A.
      • Tang J.
      • Yang X.
      • Pang Z.P.
      • Südhof T.C.
      Complexin controls the force transfer from SNARE complexes to membranes in fusion.
      ). It is interesting to note that the structures of the SNARE bundle-bound complexin CH suggest that the NTD and AH domains will be situated in the region where trans-SNARE complexes insert into the membrane. In mouse neurons, mutation of the membrane insertion sequence of synaptobrevin-2 generated a similar phenotype to that of complexin knockout as assessed by electrophysiology. Together, these observations led to the hypothesis that although these two N-terminal regions of complexin fulfill discrete facilitatory (NTD) and inhibitory (AH) functions, together they control the force transfer from SNARE complexes to membranes during fusion (
      • Maximov A.
      • Tang J.
      • Yang X.
      • Pang Z.P.
      • Südhof T.C.
      Complexin controls the force transfer from SNARE complexes to membranes in fusion.
      ). It is likely that complexin interacts with SNAREs in a variety of ways and that these varied interactions contribute to the dual functions of complexin. A recent study provided evidence for both cis- and trans-conformations of the complexin–SNARE complex. The cis-conformation, which may help to activate synchronous neurotransmitter release, required the N-terminal domain of complexin in addition to the accessory and central helices (
      • Choi U.B.
      • Zhao M.
      • Zhang Y.
      • Lai Y.
      • Brunger A.T.
      Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex.
      ).
      The first eight residues of complexin are necessary for the facilitatory function of the N-terminal domain (
      • Xue M.
      • Craig T.K.
      • Xu J.
      • Chao H.T.
      • Rizo J.
      • Rosenmund C.
      Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity.
      ). Although disordered when free in solution, the first ~17 residues of the complexin N-terminal domain appear somewhat conserved across species and, when modeled as a helix, show potentially amphipathic helical character. Methionine 5 and lysine 6 within this region are particularly critical for the facilitation of evoked release (
      • Xue M.
      • Craig T.K.
      • Xu J.
      • Chao H.T.
      • Rizo J.
      • Rosenmund C.
      Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity.
      ). This amphipathic region has been suggested to potentially bind membranes (
      • Xue M.
      • Reim K.
      • Chen X.
      • Chao H.T.
      • Deng H.
      • Rizo J.
      • Brose N.
      • Rosenmund C.
      Distinct domains of complexin I differentially regulate neurotransmitter release.
      ,
      • Maximov A.
      • Tang J.
      • Yang X.
      • Pang Z.P.
      • Südhof T.C.
      Complexin controls the force transfer from SNARE complexes to membranes in fusion.
      ,
      • Xue M.
      • Craig T.K.
      • Xu J.
      • Chao H.T.
      • Rizo J.
      • Rosenmund C.
      Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity.
      ,
      • Lai Y.
      • Choi U.B.
      • Zhang Y.
      • Zhao M.
      • Pfuetzner R.A.
      • Wang A.L.
      • Diao J.
      • Brunger A.T.
      N-terminal domain of complexin independently activates calcium-triggered fusion.
      ), although an initial NMR-based analysis showed no such NTD/membrane interaction (
      • Xue M.
      • Craig T.K.
      • Xu J.
      • Chao H.T.
      • Rizo J.
      • Rosenmund C.
      Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity.
      ). Instead, it was hypothesized that the NTD might bind to the C terminus of the SNARE complex (specifically between SNAP-25 and syntaxin-1) and, in so doing, either stabilize the SNARE complex and/or release the inhibitory function of complexin (
      • Xue M.
      • Craig T.K.
      • Xu J.
      • Chao H.T.
      • Rizo J.
      • Rosenmund C.
      Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity.
      ). Subsequent studies did, however, show a potential interaction between the N-terminal amphipathic region and lipid membranes; interestingly, NTD function did not require it to be covalently attached to the rest of complexin, and it could be functionally substituted by an unrelated hemagglutinin fusion peptide (
      • Lai Y.
      • Choi U.B.
      • Zhang Y.
      • Zhao M.
      • Pfuetzner R.A.
      • Wang A.L.
      • Diao J.
      • Brunger A.T.
      N-terminal domain of complexin independently activates calcium-triggered fusion.
      ). Ultimately, the details of how the NTD and its membrane binding contribute to complexin function remain largely unclear (Fig. 4B).
      Figure thumbnail gr4
      Figure 4Potential functions of the disordered C- and N-terminal domains of complexin. A, unstructured complexin C-terminal domain (pink) contributes to the inhibition of spontaneous synaptic vesicle exocytosis; it selectively binds to synaptic vesicles (yellow) through tandem membrane curvature-sensing motifs, termed the amphipathic helix motif (AH motif, red) and C-terminal motif (CT motif, purple). In worm complexin, the AH motif includes a π-bulge structure in the bound state, whereas the CT motif remains disordered. The AH motif also contains potential phosphorylation sites that may modulate membrane binding and thereby regulate complexin inhibitory function. Other complexin domains include the N-terminal domain (green), the accessory helix (cyan), and the central helix (orange). B, complexin N-terminal domain (green) facilitates synaptic vesicle fusion, either by binding to lipid membranes and/or by binding to the SNARE complex to displace the inhibitory accessory helix (cyan). The SNAREs, syntaxin-1, SNAP-25, and synaptobrevin-2, are shown in red, gray, and blue, respectively.
      The intrinsically disordered CTD of complexin appears necessary for the inhibition of spontaneous SV fusion in worms (
      • Martin J.A.
      • Hu Z.
      • Fenz K.M.
      • Fernandez J.
      • Dittman J.S.
      Complexin has opposite effects on two modes of synaptic vesicle fusion.
      ,
      • Wragg R.T.
      • Snead D.
      • Dong Y.
      • Ramlall T.F.
      • Menon I.
      • Bai J.
      • Eliezer D.
      • Dittman J.S.
      Synaptic vesicles position complexin to block spontaneous fusion.
      ,
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ), flies (
      • Buhl L.K.
      • Jorquera R.A.
      • Akbergenova Y.
      • Huntwork-Rodriguez S.
      • Volfson D.
      • Littleton J.T.
      Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of complexin.
      ,
      • Xue M.
      • Lin Y.Q.
      • Pan H.
      • Reim K.
      • Deng H.
      • Bellen H.J.
      • Rosenmund C.
      Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila complexins orchestrates synaptic vesicle exocytosis.
      ,
      • Cho R.W.
      • Song Y.
      • Littleton J.T.
      Comparative analysis of Drosophila and mammalian complexins as fusion clamps and facilitators of neurotransmitter release.
      • Iyer J.
      • Wahlmark C.J.
      • Kuser-Ahnert G.A.
      • Kawasaki F.
      Molecular mechanisms of COMPLEXIN fusion clamp function in synaptic exocytosis revealed in a new Drosophila mutant.
      ), mice (
      • Kaeser-Woo Y.J.
      • Yang X.
      • Südhof T.C.
      C-terminal complexin sequence is selectively required for clamping and priming but not for Ca2+ triggering of synaptic exocytosis.
      ,
      • Yang X.
      • Cao P.
      • Südhof T.C.
      Deconstructing complexin function in activating and clamping Ca2+-triggered exocytosis by comparing knockout and knockdown phenotypes.
      ), and in vitro fusion assays (
      • Lai Y.
      • Diao J.
      • Cipriano D.J.
      • Zhang Y.
      • Pfuetzner R.A.
      • Padolina M.S.
      • Brunger A.T.
      Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms.
      ): elimination or perturbation of the CTD impairs complexin inhibitory function (
      • Kaeser-Woo Y.J.
      • Yang X.
      • Südhof T.C.
      C-terminal complexin sequence is selectively required for clamping and priming but not for Ca2+ triggering of synaptic exocytosis.
      ). The CTD may also, however, have some facilitatory function, based on observations in vitro (
      • Malsam J.
      • Seiler F.
      • Schollmeier Y.
      • Rusu P.
      • Krause J.M.
      • Söllner T.H.
      The carboxy-terminal domain of complexin I stimulates liposome fusion.
      ,
      • Seiler F.
      • Malsam J.
      • Krause J.M.
      • Söllner T.H.
      A role of complexin-lipid interactions in membrane fusion.
      ), and in C. elegans (
      • Martin J.A.
      • Hu Z.
      • Fenz K.M.
      • Fernandez J.
      • Dittman J.S.
      Complexin has opposite effects on two modes of synaptic vesicle fusion.
      ). Furthermore, complexin-1 enhances the on-rate of vesicle docking through interactions of the CH with SNAREs and of the CTD with the membrane (
      • Diao J.
      • Cipriano D.J.
      • Zhao M.
      • Zhang Y.
      • Shah S.
      • Padolina M.S.
      • Pfuetzner R.A.
      • Brunger A.T.
      Complexin-1 enhances the on-rate of vesicle docking via simultaneous SNARE and membrane interactions.
      ). Notably, the aforementioned homozygous mutations to complexin observed in patients with severe infantile myoclonic epilepsy and intellectual disability could potentially perturb complexin/membrane interactions, especially given that one is a C-terminal truncation mutation. The impact of these mutations has not yet been formally assessed, however.
      The CTDs of both mouse and worm complexin have been established by NMR as intrinsically disordered (
      • Pabst S.
      • Hazzard J.W.
      • Antonin W.
      • Südhof T.C.
      • Jahn R.
      • Rizo J.
      • Fasshauer D.
      Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions.
      ,
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ), although the worm complexin CTD displays detectable residual helical structure in the free state (
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ). The CTDs of worm and mammalian complexin have been shown to interact with liposomes through a conserved amphipathic region, which in the worm protein corresponds to the location of residual helicity in the unbound state (
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ,
      • Seiler F.
      • Malsam J.
      • Krause J.M.
      • Söllner T.H.
      A role of complexin-lipid interactions in membrane fusion.
      ,
      • Gong J.
      • Lai Y.
      • Li X.
      • Wang M.
      • Leitz J.
      • Hu Y.
      • Zhang Y.
      • Choi U.B.
      • Cipriano D.
      • Pfuetzner R.A.
      • Südhof T.C.
      • Yang X.
      • Brunger A.T.
      • Diao J.
      C-terminal domain of mammalian complexin-1 localizes to highly curved membranes.
      ). The CTD/membrane interaction may contribute to inhibition and/or facilitation of synaptic vesicle fusion by the CTD. We previously established that the worm complexin CTD contains tandem lipid-binding motifs that together sense membrane curvature and selectively bind to more highly-curved membranes (
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ). A C-terminal motif rich in bulky hydrophobic residues and positively charged lysine residues initiates binding and likely remains unstructured in the membrane-bound state; an adjacent amphipathic motif adopts helical structure upon membrane binding, but only for highly-curved membrane surfaces (
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ). Mouse complexin also selectively binds to highly-curved membranes, suggesting a conserved role for membrane curvature sensing by the CTD (
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ,
      • Gong J.
      • Lai Y.
      • Li X.
      • Wang M.
      • Leitz J.
      • Hu Y.
      • Zhang Y.
      • Choi U.B.
      • Cipriano D.
      • Pfuetzner R.A.
      • Südhof T.C.
      • Yang X.
      • Brunger A.T.
      • Diao J.
      C-terminal domain of mammalian complexin-1 localizes to highly curved membranes.
      ). The structure of membrane-bound mouse complexin has not, however, been as extensively characterized. The exact mechanisms by which membrane binding might contribute to function remain unclear. We and others have proposed that the CTD localizes complexin to synaptic vesicles (Fig. 4A), an interaction that is required for inhibition of spontaneous exocytosis by complexin; this interaction would be facilitated by the preferential binding of complexin to the highly-curved synaptic vesicle membrane (
      • Wragg R.T.
      • Snead D.
      • Dong Y.
      • Ramlall T.F.
      • Menon I.
      • Bai J.
      • Eliezer D.
      • Dittman J.S.
      Synaptic vesicles position complexin to block spontaneous fusion.
      ,
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ). Additionally, for worm complexin, formation of noncanonical π-helix structure in the CTD upon membrane binding appears to be required for optimal complexin inhibitory function (
      • Snead D.
      • Wragg R.T.
      • Dittman J.S.
      • Eliezer D.
      Membrane curvature sensing by the C-terminal domain of complexin.
      ,
      • Snead D.
      • Lai A.L.
      • Wragg R.T.
      • Parisotto D.A.
      • Ramlall T.F.
      • Dittman J.S.
      • Freed J.H.
      • Eliezer D.
      Unique structural features of membrane-bound C-terminal domain motifs modulate complexin inhibitory function.
      ). This suggests a role for the CTD beyond membrane binding. It may, for example, mediate complexin protein/protein interactions, an idea supported by a recent report of potential CTD/SNARE interactions (
      • Makke M.
      • Mantero Martinez M.
      • Gaya S.
      • Schwarz Y.
      • Frisch W.
      • Silva-Bermudez L.
      • Jung M.
      • Mohrmann R.
      • Dhara M.
      • Bruns D.
      A mechanism for exocytotic arrest by the complexin C-terminus.
      ).
      Finally, the impact that CTD PTMs may have on membrane association and/or protein interactions remains essentially uncharacterized. The CTD contains potentially phosphorylatable serine and threonine residues, and mutation of human complexin serine 115–one putative site for such phosphorylation–impaired the ability of complexin to stimulate liposome fusion in an in vitro assay, although membrane binding by these mutants was not examined (
      • Malsam J.
      • Seiler F.
      • Schollmeier Y.
      • Rusu P.
      • Krause J.M.
      • Söllner T.H.
      The carboxy-terminal domain of complexin I stimulates liposome fusion.
      ). In flies, protein kinase A (PKA)-dependent phosphorylation of (fly complexin) serine 126 is required for synaptic plasticity and growth. Phosphorylation of this residue occurs as a result of retrograde signaling following stimulation, and it impairs the ability of complexin to clamp spontaneous fusion. The resultant enhanced spontaneous release then contributes to activity-dependent synaptic growth (
      • Cho R.W.
      • Buhl L.K.
      • Volfson D.
      • Tran A.
      • Li F.
      • Akbergenova Y.
      • Littleton J.T.
      Phosphorylation of complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity.
      ).
      Ultimately, a complete understanding of the function of the complexin CTD remains elusive, and the detailed mechanisms by which the CTD and CTD/membrane interactions exert their function remain unclear. Nonetheless, it provides a key example of how an intrinsically disordered region mediates a selective and functionally requisite interaction with the synaptic vesicle membrane. Future work will be required to tease out further details, including how PTMs alter CTD function and interactions. Overall, despite its small size, complexin features at least two distinct activities that are modulated in multiple ways by multiple regions, namely SNARE and synaptic vesicle binding allow complexin to clamp or facilitate exocytosis. Its intrinsically disordered character may play a key role in providing the flexibility required for carrying out opposing functions in different contexts utilizing different structures and interactions.

      α-Synuclein

      α-Synuclein is a small, soluble, and predominantly presynaptic protein that has been implicated in Parkinson's disease as well as other neurodegenerative “synucleinopathies.” Mutations in α-synuclein have been linked to familial forms of these diseases (
      • Polymeropoulos M.H.
      • Lavedan C.
      • Leroy E.
      • Ide S.E.
      • Dehejia A.
      • Dutra A.
      • Pike B.
      • Root H.
      • Rubenstein J.
      • Boyer R.
      • Stenroos E.S.
      • Chandrasekharappa S.
      • Athanassiadou A.
      • Papapetropoulos T.
      • Johnson W.G.
      • et al.
      Mutation in the α-synuclein gene identified in families with Parkinson's disease.
      • Appel-Cresswell S.
      • Vilarino-Guell C.
      • Encarnacion M.
      • Sherman H.
      • Yu I.
      • Shah B.
      • Weir D.
      • Thompson C.
      • Szu-Tu C.
      • Trinh J.
      • Aasly J.O.
      • Rajput A.
      • Rajput A.H.
      • Jon Stoessl A.
      • Farrer M.J.
      α-Synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease.
      ,
      • Chartier-Harlin M.C.
      • Kachergus J.
      • Roumier C.
      • Mouroux V.
      • Douay X.
      • Lincoln S.
      • Levecque C.
      • Larvor L.
      • Andrieux J.
      • Hulihan M.
      • Waucquier N.
      • Defebvre L.
      • Amouyel P.
      • Farrer M.
      • Destée A.
      α-Synuclein locus duplication as a cause of familial Parkinson's disease.
      ,
      • Krüger R.
      • Kuhn W.
      • Müller T.
      • Woitalla D.
      • Graeber M.
      • Kösel S.
      • Przuntek H.
      • Epplen J.T.
      • Schöls L.
      • Riess O.
      Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease.
      ,
      • Zarranz J.J.
      • Alegre J.
      • Gómez-Esteban J.C.
      • Lezcano E.
      • Ros R.
      • Ampuero I.
      • Vidal L.
      • Hoenicka J.
      • Rodriguez O.
      • Atarés B.
      • Llorens V.
      • Gomez Tortosa E.
      • del Ser T.
      • Muñoz D.G.
      • de Yebenes J.G.
      The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia.
      ,
      • Lesage S.
      • Anheim M.
      • Letournel F.
      • Bousset L.
      • Honoré A.
      • Rozas N.
      • Pieri L.
      • Madiona K.
      • Dürr A.
      • Melki R.
      • Verny C.
      • Brice A.
      French Parkinson's Disease Genetics Study Group
      G51D α-synuclein mutation causes a novel parkinsonian-pyramidal syndrome.
      ,
      • Proukakis C.
      • Dudzik C.G.
      • Brier T.
      • MacKay D.S.
      • Cooper J.M.
      • Millhauser G.L.
      • Houlden H.
      • Schapira A.H.
      A novel α-synuclein missense mutation in Parkinson disease.
      • Singleton A.B.
      • Farrer M.
      • Johnson J.
      • Singleton A.
      • Hague S.
      • Kachergus J.
      • Hulihan M.
      • Peuralinna T.
      • Dutra A.
      • Nussbaum R.
      • Lincoln S.
      • Crawley A.
      • Hanson M.
      • Maraganore D.
      • Adler C.
      • et al.
      α-Synuclein locus triplication causes Parkinson's disease.
      ), and β-sheet–rich aggregated α-synuclein can be found in Lewy bodies and Lewy neurites, two pathological hallmarks of the synucleinopathies (
      • Spillantini M.G.
      • Goedert M.
      The α-synucleinopathies: Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.
      ,
      • Spillantini M.G.
      Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are α-synucleinopathies.
      ). α-Synuclein structure, dynamics, function, and aggregation have been an extremely active area of research within the IDP field over the past 2 decades (
      • Dikiy I.
      • Eliezer D.
      Folding and misfolding of α-synuclein on membranes.
      ,
      • Snead D.
      • Eliezer D.
      α-Synuclein function and dysfunction on cellular membranes.
      ). Here, we briefly review putative synuclein functions and then focus on how IDP/membrane interactions may contribute to these.
      α-Synuclein has been implicated in synaptic plasticity (
      • Murphy D.D.
      • Rueter S.M.
      • Trojanowski J.Q.
      • Lee V.M.
      Synucleins are developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons.
      ) and learning (
      • George J.M.
      • Jin H.
      • Woods W.S.
      • Clayton D.F.
      Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.
      ), neurotransmitter release (
      • Burré J.
      • Sharma M.
      • Tsetsenis T.
      • Buchman V.
      • Etherton M.R.
      • Südhof T.C.
      α-Synuclein promotes SNARE-complex assembly in vivo and in vitro.
      ,
      • Chandra S.
      • Gallardo G.
      • Fernández-Chacón R.
      • Schlüter O.M.
      • Südhof T.C.
      α-Synuclein cooperates with CSPα in preventing neurodegeneration.
      ), and synaptic vesicle pool maintenance (
      • Murphy D.D.
      • Rueter S.M.
      • Trojanowski J.Q.
      • Lee V.M.
      Synucleins are developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons.
      ,
      • Abeliovich A.
      • Schmitz Y.
      • Fariñas I.
      • Choi-Lundberg D.
      • Ho W.H.
      • Castillo P.E.
      • Shinsky N.
      • Verdugo J.M.
      • Armanini M.
      • Ryan A.
      • Hynes M.
      • Phillips H.
      • Sulzer D.
      • Rosenthal A.
      Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system.
      ,
      • Nemani V.M.
      • Lu W.
      • Berge V.
      • Nakamura K.
      • Onoa B.
      • Lee M.K.
      • Chaudhry F.A.
      • Nicoll R.A.
      • Edwards R.H.
      Increased expression of α-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis.
      ), yet its precise function(s) remain elusive. The most widely accepted synuclein functions involve synaptic vesicle homeostasis and synaptic vesicle clustering, docking, fusion, and or recycling (
      • Dikiy I.
      • Eliezer D.
      Folding and misfolding of α-synuclein on membranes.
      ,
      • Snead D.
      • Eliezer D.
      α-Synuclein function and dysfunction on cellular membranes.
      ,
      • Burré J.
      • Sharma M.
      • Tsetsenis T.
      • Buchman V.
      • Etherton M.R.
      • Südhof T.C.
      α-Synuclein promotes SNARE-complex assembly in vivo and in vitro.
      ,
      • Nemani V.M.
      • Lu W.
      • Berge V.
      • Nakamura K.
      • Onoa B.
      • Lee M.K.
      • Chaudhry F.A.
      • Nicoll R.A.
      • Edwards R.H.
      Increased expression of α-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis.
      • Cabin D.E.
      • Shimazu K.
      • Murphy D.
      • Cole N.B.
      • Gottschalk W.
      • McIlwain K.L.
      • Orrison B.
      • Chen A.
      • Ellis C.E.
      • Paylor R.
      • Lu B.
      • Nussbaum R.L.
      Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking α-synuclein.
      ,
      • Larsen K.E.
      • Schmitz Y.
      • Troyer M.D.
      • Mosharov E.
      • Dietrich P.
      • Quazi A.Z.
      • Savalle M.
      • Nemani V.
      • Chaudhry F.A.
      • Edwards R.H.
      • Stefanis L.
      • Sulzer D.
      α-Synuclein overexpression in PC12 and chromaffin cells impairs catecholamine release by interfering with a late step in exocytosis.
      ,
      • Soper J.H.
      • Roy S.
      • Stieber A.
      • Lee E.
      • Wilson R.B.
      • Trojanowski J.Q.
      • Burd C.G.
      • Lee V.M.
      α-Synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae.
      ,
      • Eliezer D.
      ,
      • Darios F.
      • Ruipérez V.
      • López I.
      • Villanueva J.
      • Gutierrez L.M.
      • Davletov B.
      α-Synuclein sequesters arachidonic acid to modulate SNARE-mediated exocytosis.
      ,
      • Diao J.
      • Burré J.
      • Vivona S.
      • Cipriano D.J.
      • Sharma M.
      • Kyoung M.
      • Südhof T.C.
      • Brunger A.T.
      Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2.
      ,
      • DeWitt D.C.
      • Rhoades E.
      α-Synuclein can inhibit SNARE-mediated vesicle fusion through direct interactions with lipid bilayers.
      ,
      • Choi B.K.
      • Choi M.G.
      • Kim J.Y.
      • Yang Y.
      • Lai Y.
      • Kweon D.H.
      • Lee N.K.
      • Shin Y.K.
      Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking.
      ,
      • Lai Y.
      • Kim S.
      • Varkey J.
      • Lou X.
      • Song J.K.
      • Diao J.
      • Langen R.
      • Shin Y.K.
      Nonaggregated α-synuclein influences SNARE-dependent vesicle docking via membrane binding.
      ,
      • Vargas K.J.
      • Makani S.
      • Davis T.
      • Westphal C.H.
      • Castillo P.E.
      • Chandra S.S.
      Synucleins regulate the kinetics of synaptic vesicle endocytosis.
      • Ramezani M.
      • Wilkes M.M.
      • Das T.
      • Holowka D.
      • Eliezer D.
      • Baird B.
      Regulation of exocytosis and mitochondrial relocalization by α-synuclein in a mammalian cell model.
      ). It has further been suggested that synuclein may also function as a molecular chaperone for synaptobrevin-2, the synaptic v-SNARE (
      • Burré J.
      • Sharma M.
      • Tsetsenis T.
      • Buchman V.
      • Etherton M.R.
      • Südhof T.C.
      α-Synuclein promotes SNARE-complex assembly in vivo and in vitro.
      ). Other putative functions of synuclein have also been proposed, including potential roles in fatty acid and lipid metabolism (
      • Barceló-Coblijn G.
      • Golovko M.Y.
      • Weinhofer I.
      • Berger J.
      • Murphy E.J.
      Brain neutral lipids mass is increased in α-synuclein gene-ablated mice.
      • Broersen K.
      • van den Brink D.
      • Fraser G.
      • Goedert M.
      • Davletov B.
      α-Synuclein adopts an α-helical conformation in the presence of polyunsaturated fatty acids to hinder micelle formation.
      ,
      • Castagnet P.I.
      • Golovko M.Y.
      • Barceló-Coblijn G.C.
      • Nussbaum R.L.
      • Murphy E.J.
      Fatty acid incorporation is decreased in astrocytes cultured from α-synuclein gene-ablated mice.
      ,
      • De Franceschi G.
      • Frare E.
      • Bubacco L.
      • Mammi S.
      • Fontana A.
      • de Laureto P.P.
      Molecular insights into the interaction between α-synuclein and docosahexaenoic acid.
      ,
      • Golovko M.Y.
      • Faergeman N.J.
      • Cole N.B.
      • Castagnet P.I.
      • Nussbaum R.L.
      • Murphy E.J.
      α-Synuclein gene deletion decreases brain palmitate uptake and alters the palmitate metabolism in the absence of α-synuclein palmitate binding.
      ,
      • Golovko M.Y.
      • Rosenberger T.A.
      • Faergeman N.J.
      • Feddersen S.
      • Cole N.B.
      • Pribill I.
      • Berger J.
      • Nussbaum R.L.
      • Murphy E.J.
      Acyl-CoA synthetase activity links wild-type but not mutant α-synuclein to brain arachidonate metabolism.
      ,
      • Golovko M.Y.
      • Rosenberger T.A.
      • Feddersen S.
      • Faergeman N.J.
      • Murphy E.J.
      α-Synuclein gene ablation increases docosa-hexaenoic acid incorporation and turnover in brain phospholipids.
      ,
      • Karube H.
      • Sakamoto M.
      • Arawaka S.
      • Hara S.
      • Sato H.
      • Ren C.H.
      • Goto S.
      • Koyama S.
      • Wada M.
      • Kawanami T.
      • Kurita K.
      • Kato T.
      N-terminal region of α-synuclein is essential for the fatty acid-induced oligomerization of the molecules.
      ,
      • Ruipérez V.
      • Darios F.
      • Davletov B.
      α-Synuclein, lipids and Parkinson's disease.
      ,
      • Jenco J.M.
      • Rawlingson A.
      • Daniels B.
      • Morris A.J.
      Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by α- and β-synucleins.
      ,
      • Payton J.E.
      • Perrin R.J.
      • Woods W.S.
      • George J.M.
      Structural determinants of PLD2 inhibition by α-synuclein.
      ,
      • Ahn B.H.
      • Rhim H.
      • Kim S.Y.
      • Sung Y.M.
      • Lee M.Y.
      • Choi J.Y.
      • Wolozin B.
      • Chang J.S.
      • Lee Y.H.
      • Kwon T.K.
      • Chung K.C.
      • Yoon S.H.
      • Hahn S.J.
      • Kim M.S.
      • Jo Y.H.
      • Min D.S.
      α-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells.
      • Rappley I.
      • Gitler A.D.
      • Selvy P.E.
      • LaVoie M.J.
      • Levy B.D.
      • Brown H.A.
      • Lindquist S.
      • Selkoe D.J.
      Evidence that α-synuclein does not inhibit phospholipase D.
      ), dopamine synthesis and homeostasis (
      • Wersinger C.
      • Prou D.
      • Vernier P.
      • Niznik H.B.
      • Sidhu A.
      Mutations in the lipid-binding domain of α-synuclein confer overlapping, yet distinct, functional properties in the regulation of dopamine transporter activity.
      • Lee F.J.
      • Liu F.
      • Pristupa Z.B.
      • Niznik H.B.
      Direct binding and functional coupling of α-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis.
      ,
      • Perez R.G.
      • Waymire J.C.
      • Lin E.
      • Liu J.J.
      • Guo F.
      • Zigmond M.J.
      A role for α-synuclein in the regulation of dopamine biosynthesis.
      ,
      • Oaks A.W.
      • Sidhu A.
      Synuclein modulation of monoamine transporters.
      • Oaks A.W.
      • Frankfurt M.
      • Finkelstein D.I.
      • Sidhu A.
      Age-dependent effects of A53T α-synuclein on behavior and dopaminergic function.
      ), and prevention of membrane oxidation (
      • Quilty M.C.
      • King A.E.
      • Gai W.P.
      • Pountney D.L.
      • West A.K.
      • Vickers J.C.
      • Dickson T.C.
      α-Synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection.
      • Zhu M.
      • Qin Z.J.
      • Hu D.
      • Munishkina L.A.
      • Fink A.L.
      α-Synuclein can function as an antioxidant preventing oxidation of unsaturated lipid in vesicles.
      ,
      • Maltsev A.S.
      • Chen J.
      • Levine R.L.
      • Bax A.
      Site-specific interaction between α-synuclein and membranes probed by NMR-observed methionine oxidation rates.
      • Liu F.
      • Hindupur J.
      • Nguyen J.L.
      • Ruf K.J.
      • Zhu J.
      • Schieler J.L.
      • Bonham C.C.
      • Wood K.V.
      • Davisson V.J.
      • Rochet J.C.
      Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults.
      ).
      Synuclein can directly bind synaptic vesicles and appears to localize to their surface or proximity in vivo (
      • George J.M.
      • Jin H.
      • Woods W.S.
      • Clayton D.F.
      Characterization of a novel protein regulated during the critical period for song learning in the zebra finch.
      ,
      • Maroteaux L.
      • Campanelli J.T.
      • Scheller R.H.
      Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal.
      • Iwai A.
      • Masliah E.
      • Yoshimoto M.
      • Ge N.
      • Flanagan L.
      • de Silva H.A.
      • Kittel A.
      • Saitoh T.
      The precursor protein of non-A β component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system.
      ,
      • Jensen P.H.
      • Nielsen M.S.
      • Jakes R.
      • Dotti C.G.
      • Goedert M.
      Binding of α-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation.
      ,
      • Clayton D.F.
      • George J.M.
      Synucleins in synaptic plasticity and neurodegenerative disorders.
      ,
      • Kahle P.J.
      • Neumann M.
      • Ozmen L.
      • Muller V.
      • Jacobsen H.
      • Schindzielorz A.
      • Okochi M.
      • Leimer U.
      • van Der Putten H.
      • Probst A.
      • Kremmer E.
      • Kretzschmar H.A.
      • Haass C.
      Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.
      • George J.M.
      The synucleins.
      ). The N-terminal domain of α-synuclein has been clearly established as a membrane curvature sensor that folds into an amphipathic helix upon membrane binding and that displays enhanced binding to more highly-curved vesicles; binding is further enhanced by negatively charged lipids and by conical lipids (e.g. lipids with phosphatidylethanolamine headgroups) (
      • Davidson W.S.
      • Jonas A.
      • Clayton D.F.
      • George J.M.
      Stabilization of α-synuclein secondary structure upon binding to synthetic membranes.
      • Jo E.
      • McLaurin J.
      • Yip C.M.
      • St George-Hyslop P.
      • Fraser P.E.
      α-Synuclein membrane interactions and lipid specificity.
      ,
      • Rhoades E.
      • Ramlall T.F.
      • Webb W.W.
      • Eliezer D.
      Quantification of α-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy.
      ,
      • Kjaer L.
      • Giehm L.
      • Heimburg T.
      • Otzen D.
      The influence of vesicle size and composition on α-synuclein structure and stability.
      ,
      • Middleton E.R.
      • Rhoades E.
      Effects of curvature and composition on α-synuclein binding to lipid vesicles.
      • Pranke I.M.
      • Morello V.
      • Bigay J.
      • Gibson K.
      • Verbavatz J.M.
      • Antonny B.
      • Jackson C.L.
      α-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding.
      ). Synuclein thus may be optimized to bind synaptic vesicles, although it clearly interacts with other membranes as well, including possibly the plasma membrane and mitochondrial membranes (
      • Dikiy I.
      • Eliezer D.
      Folding and misfolding of α-synuclein on membranes.
      ,
      • Snead D.
      • Eliezer D.
      α-Synuclein function and dysfunction on cellular membranes.
      ,
      • Ramezani M.
      • Wilkes M.M.
      • Das T.
      • Holowka D.
      • Eliezer D.
      • Baird B.
      Regulation of exocytosis and mitochondrial relocalization by α-synuclein in a mammalian cell model.
      ). Interestingly, synuclein can, in some contexts, actively induce membrane curvature to remodel lipid membranes; this could have functional significance for synaptic vesicle exocytosis, which requires alterations in membrane shape and curvature prior to membrane fusion (
      • Bodner C.R.
      • Dobson C.M.
      • Bax A.
      Multiple tight phospholipid-binding modes of α-synuclein revealed by solution NMR spectroscopy.
      • Georgieva E.R.
      • Ramlall T.F.
      • Borbat P.P.
      • Freed J.H.
      • Eliezer D.
      The lipid-binding domain of wild type and mutant α-synuclein: compactness and interconversion between the broken and extended helix forms.
      ,
      • Varkey J.
      • Isas J.M.
      • Mizuno N.
      • Jensen M.B.
      • Bhatia V.K.
      • Jao C.C.
      • Petrlova J.
      • Voss J.C.
      • Stamou D.G.
      • Steven A.C.
      • Langen R.
      Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins.
      • Mizuno N.
      • Varkey J.
      • Kegulian N.C.
      • Hegde B.G.
      • Cheng N.
      • Langen R.
      • Steven A.C.
      Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-helical conformation.
      ).
      α-Synuclein is intrinsically unstructured in the free state (
      • Davidson W.S.
      • Jonas A.
      • Clayton D.F.
      • George J.M.
      Stabilization of α-synuclein secondary structure upon binding to synthetic membranes.
      ,
      • Weinreb P.H.
      • Zhen W.
      • Poon A.W.
      • Conway K.A.
      • Lansbury Jr., P.T.
      NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded.
      • Eliezer D.
      • Kutluay E.
      • Bussell Jr, R.
      • Browne G.
      Conformational properties of α-synuclein in its free and lipid- associated states.
      ,
      • Fauvet B.
      • Mbefo M.K.
      • Fares M.B.
      • Desobry C.
      • Michael S.
      • Ardah M.T.
      • Tsika E.
      • Coune P.
      • Prudent M.
      • Lion N.
      • Eliezer D.
      • Moore D.J.
      • Schneider B.
      • Aebischer P.
      • El-Agnaf O.M.
      • et al.
      α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer.
      • Theillet F.X.
      • Binolfi A.
      • Bekei B.
      • Martorana A.
      • Rose H.M.
      • Stuiver M.
      • Verzini S.
      • Lorenz D.
      • van Rossum M.
      • Goldfarb D.
      • Selenko P.
      Structural disorder of monomeric α-synuclein persists in mammalian cells.
      ). Its N-terminal ~100 residues constitute a lipid-binding domain (Fig. 5A) featuring seven imperfect 11-residue repeats that can adopt an amphipathic helical structure upon binding to detergent micelles or phospholipid vesicles (
      • Dikiy I.
      • Eliezer D.
      Folding and misfolding of α-synuclein on membranes.
      ,
      • Snead D.
      • Eliezer D.
      α-Synuclein function and dysfunction on cellular membranes.
      ,
      • Bussell Jr., R.
      • Eliezer D.
      A structural and functional role for 11-mer repeats in α-synuclein and other exchangeable lipid binding proteins.
      ,
      • Chandra S.
      • Chen X.
      • Rizo J.
      • Jahn R.
      • Südhof T.C.
      A broken α-helix in folded α-synuclein.
      ). The very N terminus of free N-terminally acetylated α-synuclein does exhibit some residual helical structure, as evidenced by NMR-derived carbon secondary shifts (
      • Kang L.
      • Moriarty G.M.
      • Woods L.A.
      • Ashcroft A.E.
      • Radford S.E.
      • Baum J.
      N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer.
      • Fauvet B.
      • Fares M.B.
      • Samuel F.
      • Dikiy I.
      • Tandon A.
      • Eliezer D.
      • Lashuel H.A.
      Characterization of semisynthetic and naturally Nα-acetylated α-synuclein in vitro and in intact cells: implications for aggregation and cellular properties of α-synuclein.
      ,
      • Maltsev A.S.
      • Ying J.
      • Bax A.
      Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties.
      • Dikiy I.
      • Eliezer D.
      N-terminal acetylation stabilizes N-terminal helicity in lipid- and micelle-bound α-synuclein and increases its affinity for physiological membranes.
      ). This transient helical structure appears to facilitate membrane binding (
      • Dikiy I.
      • Eliezer D.
      N-terminal acetylation stabilizes N-terminal helicity in lipid- and micelle-bound α-synuclein and increases its affinity for physiological membranes.
      ) and can be considered to be a PreSMo or MoRF.
      Figure thumbnail gr5
      Figure 5Conformational plasticity of α-synuclein. α-Synuclein is intrinsically disordered in the free state, adopts helical structure upon membrane binding, and can aggregate into β-sheet–rich amyloid fibrils. A, N-terminal ~100 residues constitute the membrane-binding domain of α-synuclein and can be subdivided into helix 1 (green) and helix 2 (purple) based on its micelle-binding properties. Helix 2 contains the hydrophobic NAC region (cyan) that is believed to drive α-synuclein aggregation. The C-terminal ~40 residues form an acidic C-terminal domain (red). Phosphorylation sites discussed in the text are indicated. B, membrane-bound α-synuclein can adopt either an extended-helix or a broken-helix conformation with helices 1 and 2 separated by a short linker region; the broken-helix conformation may allow synuclein to bridge discrete membranes and so to function in vesicle docking and/or vesicle clustering. C, α-synuclein can bind to membranes via helix 1 alone. This binding mode may facilitate aggregation of the adjacent hydrophobic NAC region by promoting intermolecular interactions on the membrane surface.
      Once membrane binding is initiated at the N-terminal end of α-synuclein, coupled membrane binding and folding propagates the initial helical structure through the remainder of the N-terminal lipid-binding domain. Several different membrane-bound helical conformations have been reported featuring amphipathic helices of varying lengths that lie along the surface of the membrane. In the extended-helix conformation, the entire N-terminal domain (~100 residues) binds to the membrane surface through a continuous amphipathic α-helix with an unusual 11/3 periodicity (wherein 11 residues form 3 helical turns, a slightly overwound geometry compared with α-helices, where 18 residues form 5 turns) (
      • Georgieva E.R.
      • Ramlall T.F.
      • Borbat P.P.
      • Freed J.H.
      • Eliezer D.
      The lipid-binding domain of wild type and mutant α-synuclein: compactness and interconversion between the broken and extended helix forms.
      ,
      • Bussell Jr., R.
      • Eliezer D.
      A structural and functional role for 11-mer repeats in α-synuclein and other exchangeable lipid binding proteins.
      ,
      • Jao C.C.
      • Der-Sarkissian A.
      • Chen J.
      • Langen R.
      Structure of membrane-bound α-synuclein studied by site-directed spin labeling.
      • Bussell Jr., R.
      • Ramlall T.F.
      • Eliezer D.
      Helix periodicity, topology, and dynamics of membrane-associated α-synuclein.
      ,
      • Borbat P.
      • Ramlall T.F.
      • Freed J.H.
      • Eliezer D.
      Inter-helix distances in lysophospholipid micelle-bound α-synuclein from pulsed ESR measurements.
      ,
      • Georgieva E.R.
      • Ramlall T.F.
      • Borbat P.P.
      • Freed J.H.
      • Eliezer D.
      Membrane-bound α-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.
      ,