Sunday, 27 May 2012

Lecture material: Structural insights into the molecular mechanism of calcium-dependent vesicle–membrane fusion by Brunger


Fig 1 life cycle of a synaptic vesicle

Introduction

  • Vesicular trafficking in eukaryotic cells is essential for diverse cellular processes, including maintenance of distinct subcellular compartments, protein and hormone secretion, egg fertilization and neurotransmitter release
  • the life cycle of a vesicle generally consists of three stages (Fig. 1)
  1. endocytosis or formation of the vesicle from specific cellular membranes
  2. exocytosis or fusion of the vesicle with its target membran
  3. recycling of the components of the protein machinery after exocytosis. 
  • In synaptic vesicle exocytosis, three SNARE proteins are involved: the plasma-membrane-associated proteins syntaxin and SNAP-25 (synaptosomal-associated protein of 25kDa), and the vesicular protein synaptobrevin, also referred to as VAMP (vesicle-associated membrane protein).
  • Other conserved proteins include the ATPase NSF and its adaptor, known as SNAP (soluble NSF-attachment protein), the Rab class of small G proteins and their effectors, the synaptotagmin family and the nSec1 (neuronal homolog of the yeast Sec1 protein, also referred to as Munc18) family.

Fig. 2. Stages involved in vesicle–membrane fusion and key proteins involved. The proteins in the central schematic are color coded as follows: synaptobrevin, dark blue; synaptophysin, light blue; syntaxin, red; nSec1, brown; SNAP-25, green; synaptotagmin, yellow; Rab3A, dark red circle; rabphilin-3A, olive green; Ca2+ channel, magenta; NSF, pink circle; α-SNAP, sky blue. Surrounding the schematic are crystal structures of the SNARE complex (blue: synaptobrevin; red: syntaxin; green: SNAP-25) [29], the N-terminal domain of syntaxin, which is shown as a separate structure (the structure of the linker between the syntaxin N-terminal domain and the core SNARE complex is unknown) [30], the nSec1–syntaxin complex (red: syntaxin; brown: nSec1) [38••], α-SNAP (Sec17 in yeast) [110••], NSF-N[106••.] and [107••.], NSF-D2 [103.] and [104.], the complex between the small G protein Rab3A and the effector binding domain of rabphilin-3A (red: Rab3A; brown: rabphilin-3A) [92••], Rab GDI [96] and domains C2A and C2B of synaptotagmin [72••]. Pi, inorganic phosphate.
  •  Initially, syntaxin is bound to nSec1 and synaptobrevin is probably bound to a factor such as synaptophysin or VAP33.
  •  yet to be identified molecular machinery probably brings the vesicle and plasma membrane into close proximity
  • lets SNAREs on opposite membranes form cis complexes.
  • Synaptobrevin then binds to syntaxin and SNAP-25
  • At the priming stage, the system becomes competent to undergo fusion upon an increase in Ca2+ concentration in the micromolar range
  • possibly involving a Ca2+-binding protein such as synaptotagmin
  • At the recycling stage, α-SNAP and NSF bind to the SNARE complex, 
  • SNARE complex is then dissociated upon ATP hydrolysis.
  • Before docking, vesicles have to be targeted to the correct location at the appropriate time.
Fig. 3. Known crystal structures of components of the 20S complex — SNARE complex [29], α-SNAP (Sec17 in yeast)[110••], NSF-N [106••.] and [107••.] and NSF-D2 [103.] and [104.] — and their speculative localization in a rotational averaged electron micrograph of the 20S complex [102]. The packing of the NSF-D2 domain in the crystallographic P6 lattice forms a hexamer [103.] and [104.] that matches the cone-shaped ring-like features of the electron micrograph. As the D1 and D2 domains have similar primary sequences, their structures are probably also similar. This suggests that the two rings consist of the D1 and D2 domains. The assignment of the N-terminal domain was suggested by matching the trimeric packing of the three NSF-N domains per asymmetric unit in one of the crystal forms [107••] with the electron micrograph.

SNAREs

  • The SNARE core complex consists of a parallel four-helix bundle
  • N-terminal domain of syntaxin consists of an antiparallel three-helix bundle
  • the core of the four-helix bundle of the SNARE complex is composed of layers formed by interacting sidechains from each of the four α helices
  • At centre of core complex, cnoserved ionic layer
  • has arginine and 3 glutamines contributed by each of 4 alpha helices.
  • Ionic layer is sealed off against solvent by adjacent hydrophobic layers.
  • Mutations in these and other layers reduce complex stability
  • cause defects in membrane trafficking
Fig. 4. Conformational states and binding events involving SNARE proteins and their possible role in vesicle fusion (zipper model). As discussed in more detail in the text, SNAREs have several conformational states: (a) closed; (b)binary (syntaxin–SNAP-25); and (c,d) ternary cis SNARE complex. The intermediate cis complex shown in (c) is speculative. Synaptobrevin, blue; syntaxin, red; SNAP-25, green. Undetermined: no information available about the conformation of the protein region; flexible, residues that are probably undergoing significant motion in solution and are not part of a rigid domain of the protein. TM, transmembrane.
  • SNAREs have several conformational states
  1. Closed conformation of uncomplexed syntaxin and unstructured or flexible comforms of synaptobrevin and SNAP-25
  2. Binary complex of syntaxin and SNAP-25
  3. Ternary complex of syntaxin, SNAP-25 and cytoplasmic domain of synaptobrevin
  • Closed conformation of uncomplexed syntaxin contains a 4-helix bundle 
  • bundle made up of regulatory Nterminal  HAHBHC domain and roughly half of the core complex domain Hcore
  • A similar conformation of syntaxin has recently been observed in the crystal structure of syntaxin in the syntaxin–nSec1 complex
  •  may be the closed conformation of syntaxin that binds to nSec1.
  • Syntaxin switches to an ‘open’ state upon binding to SNAP-25
  •  In this ‘open’ state, binding to the other SNAREs is mediated by the Hcore domain

Interactions of syntaxin with nSec1

  • Partially structured, closed state of syntaxin interacts with nSec1.
  • When complexed with nSec1, C terminal syntaxin residues unstructured or flexible in solution adopt a sequence of small a-helical frags connected by short loops
  • In ternary SNARE complex these residues form a continuous a helix. 
Synaptotagmin
  • Synaptotagmin is a membrane-associated protein that interacts with SNAREs, phospholipid membranes, Ca2+ channels and proteins involved in endocytosis
  • In cytosolic portion, a flexible seven aa linkter joins 2 honomologous C2 domains, C2A and C2B.
  • The C2A domain binds to anionic phospholipids and other accessory proteins, such as syntaxin
  • in a Ca2+-dependent fashion.
  • No conformational change is observed upon Ca2+ binding 
  • except for rotamer changes of the Ca2+-coordinating aspartic acid residues. 
  • The C2B domain promotes binding to other C2B domains as well as to accessory proteins, independently of Ca2+
  • Crystal structure of synaptotagmin II includes C2A and C2B domain
  • Has differences in shape of Ca2+ binding pocket, electrostatic surface potential and stoichiometry of bound divalent cations for 2 domains
  • Synaptotagmin covalently links 2 inndependent C2 domains, each with potentially different binding partners
  • C2B is involved in synaptotagmin oligomerisation
  • Synaptotagmin and SNARE complex interact indepent of Ca2+
  • Ca2+ enhances interaction
  • Ca2+ binding domains interact with plasma membrane

NSF

  • Model says, NSF and SNAP family members act together to disassemble SNARE complexes before and after fusion
  • SNARE proteins can form cis (same mebrane) and tarns (opposing membrane) complexes
  • complexes are substrates for SNAPs and NSF
  • Trans SNARE complexes important for membrane fusion
  • results in cis SNARE complex formation
  • they are dissasembled 
          --- recycling and reactivation by SNAP and NSF
  • NSF interacts with glutamate receptors
  • NF helps glutamate receptor cycling in and out of synaptic postsynaptic membrane
  • thru endocutosis and exocytosis
  • NSF is a hexamer
  • belongs to AAA family (ATPases assoc with cell activities)
  • Each NSF protomer has 3 domains
  1. N terminal domain for SNAP-SNARE binding
  2. 2 ATPase domains, D1 and D2
  • ATP binding and hydrolysis by  D1 are necessary for SNARE dissasembly
  • ATP binding (not hydrolysis) by D2 necessary for hexamer formation
  • SNAP and NSF bind sequentially to SNARE complexes
          --- form 20S particles

NSF-D2

  • NSF--D2 consists of a nt-binding subdomain and a C-terminal subdomain
  • There are interactions between bound ATP moietry and neighbouring D2 protomer and C terminal subdomain
  • may be important for ATP-dependent oligomerisation
  • Conserved lysines interact with ATP β- and γ-phosphates
  • one of which emerges from a neighboring NSF protomer 
  • probably contributes to the low hydrolytic activity of D2

NSF-N

  • N terminal domain of NSF required for SNAP-SNARE binding and disassembly.
  • The N-terminal domain is composed of two subdomains: a double-ψ−ψ-barrel and an α−β roll
  • Interface between subdomains forms a groove
  • probable site of interaction with C-terminal portion of a-SNAP
  • Both subdomains are structurally similar to domains of TF, EF-Tu. 
  • Both proteins have adj nt-binding domain D1 in NSF and domain 1 in EF_Tu
  • Both proteisn couple nt hydrolysis to large conform change between domains.

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