Intrinsically disordered proteins in synaptic vesicle trafficking and release

Abstract

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.

Keywords: complexin; intrinsically disordered protein; lipid vesicle; membrane trafficking; membraneless organelles; neurotransmitter release; phase transitions; protein folding; synaptic vesicles; α-synuclein.

Figure 1.

Intrinsically 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.

Figure 2.

SNARE 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.

Figure 3.

Functional 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).

Figure 4.

Potential 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.

Figure 5.

Conformational 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.