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.

Saturday, 26 May 2012

Lecture materials: Molecular architecture of native HIV-1 gp120 trimers by Jun Liu



FIGURE 4. Description of the conformational change in the gp120 trimer induced by CD4 binding.ad, Model for the conformational change from the unliganded (ac) to the CD4-bound state (bd) shown as top (ab) and front (cd) views. The gp120 core, CD4, V1/V2 and V3 stems are shown in white, yellow, red and green colours, respectively. e, Schematic description of the gp41 (blue) and gp120 (red/purple) regions of the trimeric spike and the conformational changes that occur upon CD4 binding. The yellow patch near the apex marks the location of the CD4 binding site in the unliganded spike and the green patch at the apex marks the location of the V3 loop region in the spike after CD4 binding. f, Schematic view of the consequence of the CD4-induced conformational changes for viral attachment to the target cell and interaction with chemokine receptors (green at top). Colours in f have same meaning as in e.


  • CD4 induces opening of trimer
  • CD4 binding contributes to entropy
  • Opening of trimer makes way for exposure of central gp 41 stalk.
  • The V3 loop region is released from the lateral edge of the apex of the spike to directly point towards the target cell
  • while the V1/V2 regions as well as the CD4 binding sites move away from the centre of the spike (Fig. 4b).
  • In native state, trimer is held together by strong contacts at gp 41 base and apex.
  • Little contact between other regions of neighbouring gp120 monomers
  • cause spike archituecture held together
  • sprung open on CD 4 binding
  • The outward movement of gp120 results in a steep change in the orientation of the two outermost domains (D1D2) of CD4 (Fig. 4e),
  • must draw the virus closer to the target cell membrane by virtue of the flexibility between the D1D2 and D3D4 domains of membrane-anchored CD4 (Fig. 4f). 

Lecture material; Viral membrane fusion by Stephen C Harrison

  • Fusion of 2 bilayer membranes is thermodynamically favourable
  • High kinetic barrier.
  • Fusogens lower barrier
  • Viral proteins lower barrier by using free enerby liberated during protein conform. change to draw membranes together.
  • Hemifusion state: apposed proximal leaflets of 2 bilayers but not distal leaflets have merged is obligatory intermediate. 
  • Evidence suggests viral fusion proteins lower kinetic barriers and catalyse membrane fusion. 

(a) The protein in the pre-fusion conformation, with its fusion peptide or loop (light green) sequestered. The representation is purely schematic, and various features of specific proteins are not incorporated—for example, the displacement of the N-terminal fragment of proteins that are cleaved from a precursor or the dimer-to-trimer rearrangement on the surface of flaviviruses. (b) Extended intermediate. The protein opens up, extending the fusion peptide or loop to interact with the target bilayer. The part of the protein that bears the fusion peptide forms a trimer cluster. (c) Collapse of the extended intermediate: a C-terminal segment of the protein folds back along the outside of the trimer core. The segments from the three subunits fold back independently, so that at any point in the process they can extend to different distances along the trimer axis, and the entire trimer can bow outward, away from the deforming membrane. (d) Hemifusion. When collapse of the intermediate has proceeded far enough to bring the two bilayers into contact, the apposed, proximal leaflets merge into a hemifusion stalk. (e) Fusion pore formation. As the hemifused bilayers open into a fusion pore, the final zipping up of the C-terminal ectodomain segments snaps the refolded trimer into its fully symmetric, post-fusion conformation, preventing the pore from resealing.
Step 1
  • Protein opens up
  • forms a bridge between 2 bilayers
  • All viral fusion proteins studied have 2 membrane-interacting elements:
  1. a C-terminal TM anchor that holds protein in viral membrane
  2. a hydrophobic patch (fusion peptide or fusion loop_ that interacts with target membrane. 
  • All are trimeric in fusion-active state.
  • When ligand binds (eg protons, as mech responds to low pH of endosome, cell or viral protein ligand)
  • fusion protein undergoes conform change
  • extend each subunit towards target membrane
  • yield contact between membrane and fusion peptide or loop
  • many fusion proteins are C-terminal frags of larger precursor (eg HA2 fram of influenza virus hemaglutinin or gp41 frag of HIV Env)
  • To initiate fusion they must shed N-terminal frag
  • N-terminal frag has receptor-binding domain (HA1 or gp120)
  • Extended state is pre-hairpin intermediate
  • Collapse into folded back conform.
  • HIV-1 gp41 intermediate has half life of many minutes
  • for others may be few seconds

.Step 2
  • The bridge collapses. 
  • fusion peptide or loop in target membrane and C-terminal TM anchor in viral membrane come together.
  • Collapse distorts 2 bilayers 
  • into nipplelike conformation.
Step 3
  • Distortion of membranes lowers energy barrier between separated and hemifused membranes 
  • hemifused stalk forms


 Step 4
  • Hemifusion stalk opens to form a transient fusion pore.
  • Final conformational step in protein refolding renders open state irreversible
  • Pore expands.
  • With some fusion proteins, pore may flicker open and close
  • Steps 3 and 4 probably need concerted action of more than one fusion protein trimer.
  • 3 viral fusion proteins from influenza, dengue, and vesicular stomatitis viruses are from class I, II and III viral fusion proteins. 


Fig 2: Free energy changes during fusion of bilayers. The relative heights of the various barriers are arbitrary. Fusion proteins accelerate the process by coupling traversal of these barriers to energetically favorable conformational changes.   

Influenza virus hemagglutinin
  • Core of HA1 is a sialic acid-binding domain borne on stalk formed by HA2
  • Central feature of stalk is a 3-chain, α-helical coiled coil.
  • HA0 and its cleaved product, prefusion HA1-HA2 are identical ins overall structure.
  • Cleavage occus in trans Golgi network and after viral budding
  • Cleavage causes modest local rearrangement
  • newly generate N terminus of HA2 inserts into a pocket along threefold axis.
  • Buries fusion peptide. 
  • Pocket is created when C termini of HA2 coiled-coil helices splay apart
  • 3 helices diverge from threefold axis and from each other like tripod (fig 3a)
  • Sialic acid on glycoproteins or glycolipds is influenza virus receptor
  • HA1 bears binding site
  • Shallow pocket exposed on its outward-facing surface. 
  • Plasma membrane recycles regularly through endocytosis
  • virus-receptor complex may not need a spec endocytic signal to reach an endosome.
  • HA1:HA2 trimer encounters low p ---> undergoes conform. rearrangement ---> HA1 separates from HA2 (3a to 3b) except for a residual disulphide tether
  • Latter turns inside out.
  • In HA2 folding, loop to helix transition 
  • In region connecting fusion peptide to central coiled coil (3b to 3c)
  • C terminal of molecule reorients so it zips up alongside extended coil (3c to 3d)
  • Correspond to formation of extended intermediate and collapse into a conform that brings together fusion peptide and TM anchor.
  • Loop to helix transition in N terminal part of HA2 augments central coiled coil at N terminal end (3c)
  • C terminal part of protein reorients ---> break central helices where they splay apart
  • at site from which fusion protein withdrew earlier in fusion process.
  • Mutations affect stability of trimer interfaces that break during rearrangement
  • alter threshold pH for fusion
  • Conserved ionisable residues (2 Asp and a His) in vicinity of buried fusion protein may contribute to trigger
  • Fusion peptide is thought to form an amphipathic helix
  • When fusion is complete, pp chain segment C terminal to fusion peptide and membrane-proximal segment of HA2 interact
  • Postfusion comform of HA2 : residues connecting fusion peptides to coiled voil and residues N terminal to TM anchors cap the N termini of 3 central helices
  • Extended intermediate collapses
  • It must bend outwards away from nascent hemifusion stalk
  • let 2 membranes come together. 
  • Each HA2 chain can complete refolding independently of other 2
  • Loss of 3 fold symmetry
  • Forming cap restores global 3 fold symmetry (3e)
  • If TM helices pass completely thru bilayer, may need presence of aqueous channel - a committed fusion pore

Figure 3 - Influenza virus hemagglutinin: proposed sequence of fusogenic conformational changes.

Activating and initiating

  • Priming is result of proteolysis
  • Either of fusion protein (influenza hemagglutinin or retroviral Env) or an accompanying protein e.g. flavivirus and alphavirus.
  • For these viruses, priming occurs during transport of immature glycoprotein to cell surface
  • either before assmebly of virus aprticle by budding at cell surface
  • or after formation of an immature particle by budding thru an internal membrane
  • Glycoproteins of other viruses eg Ebola and SARS coronavirus need to be cleaved byendosomal cathepsins B or L during cell entry rather than during maturation 
  • Primed fusion protein is metastable in prefusion state.
  • Covalent peptide bond in fusion protein, or chaperone restrains initial folded conform of precursor.
  • After covalent restraint is gone high kinetic barrier separates primed from postfusion conform.
  • To lower barrier can be proton binding for viruses that evolved to detect low pH endosomal env
  • or binding of a coreceptor in HIV-1.
  • For herpesviruses and paramyxoviruses, trigger is altered lateral contact with other viral surface protein
  • Association with ligand alters free energy profile
  • so rearrangement to postfusion state is rapid.
  • Free energy liberated by rearrangement can overcome barrier to merging 2 membrane bilayers.
  • Extended intermediate forms.

The extended intermediate

  • Post fusion conform of gp41 ectodomain is a trimer of hairpins
  • both prongs of hairpin are a-helices. 
  • Approximately 50 residues immediately C-terminal to the fusion peptide (designated HR1, where "HR" stands for "heptad repeats") form a central, three-chain coiled coil.
  •  A loop that contains a conserved disulfide bond connects the HR1 segment to a second heptad-repeat element, HR2, which forms an outer-layer alpha-helix. 
  • Peptides from this outer layer can inhibit the fusion process
  • The mechanism involves association of the peptide with the inner core,
  • preventing transition to the post-fusion conformation.
  •  HIV fusion occurs at the cell surface, and one such inhibitory peptide (T-20, or enfuvirtide) is a clinically useful drug 
  • Mutations conferring resistance to T-20 can occur at various positions in the envelope protein, including residues in gp120 
  •  Some of the mutations in HR1 that reduce T-20 binding also retard fusion and enhance sensitivity to antibodies targeting the membrane-proximal region of gp41 
How many fusion-protein trimers contribute to formation of a fusion pore?
  • The energy barrier that must be overcome en route to a hemifusion stalk is thought to be about approx40–50 kcal mol-1 
  • 15-20 fusion proteins are estimated for HIV. 
  • In a mechanism of cooperativity, lateral contacts form between adj trimers in a ring round fusion pore site.
  • Proposed for alphavirus. 
  • Lateral contacts couple conform. change of one trimer to that in another, like allosteric regulation of miultisubunit enzymes. 
  • Other mechanism caused by response of 2 membranes to distortions necessary to promote fusion. 
  • 2 properties of bilayer membranes cause them to resist collapse of extended intermediate.

  1. Energy of bending in bilayer membranes into nipple like configuration.
  2. Hydration force creates barrier when apposing membranes come closer than  10–20 Å 
  • All proteins in fusion event bridge same pair of membranes
  • So behavious of one extended intermediate is not independent of behaviour of neighbouring one
  • They are coupled by deformation energies of 2 bilayers they connect.
  • Deforming a planar membrane into a bed creates a cap with positive curvature and a flared reigion joining it to planar membrane of the membrane. 
  • Fusion loops and peptides insert only partway into outer leaflet of traget membrane
  • displace lipid head groups laterally
  • favours curvature of leaflet. 
Does the membrane-proximal segment of the ectodomain have a mechanistic role?

  • The membrane-proximal segments (10–15 residues) of many fusion-protein ectodomains have characteristic hydrophobic and often relatively tryptophan-rich sequences.
  • At the end of the fusion-inducing conformational change, these segments are apposed to the merged membrane and could in principle interact with the membrane or the fusion peptides/loops (or both).
What structural rearrangements lower the kinetic barrier between hemifusion and fusion-pore formation? 

  • If influenza virus hemagglutinin is liked to  a glycosyl phosphatidylinositol anchor or to a truncated protein anchor that does not completely traverse the membrane, the fusion process halts at hemifusion and proceeds forward very slowly
  • Collapse of extended intermediate may not be enough to drive fusion process to completion.
  • At hemifusion stage, a hemagglutinin-induced fusion pore can flicker open and close.
  • Central helix in influenza HA2 (fig 3e) caps
  • shows formation of structure with tight hydrophobic and hydrogen-bonding interactions among aa
  • brings about concerted closure of conformational transition
  • draw near ends of TM helices together
  • cytoplasmic segments on opposite side of membrane pass into or thru nascent fusion pore.
  • Segments in aqueous channel of fusoin pore prevent its resealing
  • formation of cap on HA2 helical bundle when zipping up is complete makes pore formation irreversible.
  • cap formation couples to a translocation, into or through the pore, of material from the cytoplasmic face of the bilayer.

Fusion inhibitors

  • Ligands can retard or block viral entry if they bind selectively to any conformation of the fusion protein that precedes merger of the two bilayers.
  • Some neutralising antibodies block viral infection by inhibiting fusion
  • Some small molecules bind HIV gp 120 in prefusion conformation (as in HIV-1)
  • raise barrier to initial steps of fusion sequence
  • Site for molecules may be pocket in gp 120 
  • closes up in initial CD4-induced conformation transition
  • Productive receptor binding must expel compound from site and incur inhibitor-spec free energy cost.
  • A similar site on dengue virus E is a pocket that disappears when protein starts to rearrange 

Friday, 25 May 2012

Lecture material: Structure of the dengue virus envelope protein after membrane fusion


  • Membrane fusion is the central molecular event during the entry of enveloped viruses into cells. 
  • Viral surface proteins facilitate bilayer fusion
  • They are triggered by viral interaction with target cell.
  • The best-studied example is the influenza virus haemagglutinin (HA),
  • HA is synthesized as a single-chain precursor
  • It is then cleaved into two chains, known as HA1and HA2, during transport of the trimeric glycoprotein to the cell surface. 
  • The binding of HA1 to a cell-surface receptor leads to endocytic uptake
  • acidification of the endosome triggers dramatic conformational rearrangement of HA2
  • The latter is a two-stage process.
  •  Exposure of the aminoterminal ‘fusion peptide’ of HA2
  •  allows it to insert into the endosomal membrane
  • Whole HA2 pp chain subsequently folds over
  • This brings together its N and C termini  forces the target-cell membrane (held by the fusion peptide) and theviral membrane (held by the C-terminal transmembrane anchor ofHA) against each other.
  • HA is the prototype of a large class of viral fusion proteins—for example, those of other myxo- and paramyxoviruses such as measles virus, retroviruses such as HIV, and filoviruses such as Ebola virus
  • All of these ‘class I’ viral fusion proteins are two-chain products of a cleaved, single-chain precursor, 
  • All bear a hydrophobic fusion peptide at or near the N terminus created by the cleavage 
  • in all class I fusion proteins, a three-chain, a-helical, coiled-coil assembles during the conformational change
  • this drives the fusion peptide towards the target-cell membrane
  • This creates central structural element of the fusion machinery.
  • An architecturally and evolutionarily distinct class of fusion proteins is found on flaviviruses, such as yellow fever, West Nile,and dengue viruses, and on alphaviruses, such as Semliki Forest and Sindbis viruses.
  • These proteins associate with a second, ‘protector’ protein, 
  • the cleavage of protector protein primes the fusion protein to respond to acidic pH.
  • Structures have been determined for the ectodomains of three class II proteins in their prefusion state
  • Those of two flaviviruses, tick-borne encephalitis (TBE) and dengue viruses, are dimeric, both in solution and on the viral membrane surface
  • They have three domains, with folds based largely on b-sheets. 
  • One of these (domain II), an elongated, finger-likestructure, bears a loop at its tip with a hydrophobic sequence conserved among all flaviviruses. 
  • Experiments with TBE virus show that this ‘cd loop’ (residues 98–109 in dengue type 2) is responsible for attachment of soluble E ectodomains to target membranes 
  • The hydrophobic residues are essential for its activity
  • cd loop of class II fusion proteins has a function analogous to that of the N-terminal fusion peptide in class I fusion proteins: 
  1. insertion into the host-cell membrane
  2. provision of an attachment point for drawing host-cell and viral membranes together
  • We refer to the cd loop as the ‘fusion loop’, reserving ‘fusion peptide’ for the N-terminal segment of class I fusion proteins
  • In dengue virus type 2 protein (sE) in its trimeric, postfusion conformation,
  • The fusion loops of the three subunits come together to form a membrane-insertable,‘aromatic anchor’ at the tip of the trimer. 
  • The fusion loop retains its prefusion conformation. 
  • Neighbouring hydrophilic groups restrictinsertion to the proximal part of the outer lipid-bilayer leaflet.
  • The entire ectodomain of the protein folds back on itself,
  • This directs the C-terminal, viral membrane anchor towards the fusion loop
Membrane insertion and trimer formation
  • Dimer formed by dengue sE dissociates reversibly.
  • At acidic pH, dissociation is essentially complete at protein concentrations of 1 mg ml
  • at neutral pH, the dissociation constant is one to two orders of magnitude smaller. 
  • The fusion loop at the tip of domain II would be exposed in the monomer
  • but exposure does not cause nonspecific aggregation of the protein
  • experiments show that the fusion loop of monomeric TBE sE allows association with lipid membranes and that this membrane association catalyses irreversible formation of sE trimers at low pH 
  • on acidification, sE dimers dissociate, bind liposomes and trimerize 
  • The trimers are tapered rods, about 70–80 A˚long and 30–50 A˚in diameter, with the long axis perpendicular to the membrane and their wide end distal to it. 
  • They tend to cluster on the liposome surface, often forming a continuous layer. 
  • These heavily decorated areas appear to have a greater than average membrane curvature, resulting in smaller vesicles
  • This observation suggests that E trimers can induce curvature, a property that may help promote fusion
  • The dengue sE trimers can be solubilized with the detergent n-octyl-b-D-glucoside (b-OG); they remain trimeric at all pH values between 5 and 9, as determined by gel filtration chromatography 
The fusion loop
  •  the fusion loop is stable when fully exposed. 
  • It thus appears that the fusion loop retains essentially the same conformation, whether buried against another subunit, inserted into a lipid membrane, or exposed to aqueous solvent.
  • In the trimer, the three hydrophobic residues in the fusion loop conserved among all flaviviruses—Trp 101, Leu 107 and Phe 108—are fully exposed on the molecular surface, near the three-fold axis.
  • They form a bowl-like concavity at the trimer tip, with a hydrophobic rim 
  • Tryptophans tend to appear in membrane proteins at the interface between the hydrocarbon and head-group layers of the lipid
  • if the indole amine participates in a hydrogen bond, as is the case for Trp 101, the side chain may be completely buried in the hydrocarbon layer.
  • We therefore propose that the E trimers penetrate about 6 A˚ into the hydrocarbon layer of the target membrane. 
  • They cannot penetrate further, because of exposed carbonyls and charged residues on the outside rim of the fusion-loop bowl
  • the fusion loop is held in the membrane mainly by an ‘aromatic anchor’ formed by Trp 101 and Phe 108. 
  • The bowl is lined by the hydrophobic side chains of Leu 107 and Phe 108, so that it cannot accommodate lipid headgroups
  • fatty-acid chains from the inner leaflet of the membrane may extend across to contact the base of the fusion-loop bowl, or that fatty-acid chains from the outer leaflet may bend over to fill it. 
  • In either case, insertion will produce a distortion in the bilayer
A postfusion conformation
  • Domain III folds back
  • B-strands rearrange at trimer interface
  • This projects C terminus if sE towards fusion loop
  • Position fusion loop at entrance of a channel which extends towards fusion loops
  • Stem connecting end of sE fragment with viral TM anchor could span length of channel
  • By binding channel, stem would contribute more trimer contacts with domain II of another subunit. 
  • Proposed stem conformation puts viral TM domain in vicinity of fusion loop
  • Like postfusion conformation of class I viral fusion proteins.
Propsed mechanism for fusion mediated by class II viral fusion proteins. Full-length E is represented as in Fig. 1c, with the stem and viral transmembrane anchor in cyan. a, E binds to a receptor on the cell surface and the virion is internalized to an endosome. b, Reduced pH in the endosome causes domain II to hinge outward from the virion surface, exposing the fusion loop, and allowing E monomers to rearrange laterally in the plane of the viral membrane. c, The fusion loop inserts into the hydrocarbon layer of the host-cell membrane, promoting trimer formation. d, Formation of trimer contacts spreads from the fusion loop at the tip of the trimer, to the base of the trimer. Domain III shifts and rotates to create trimer contacts, causing the C-terminal portion of E to fold back towards the fusion loop. Energy release by this refolding bends the apposed membranes. e, Creation of additional trimer contacts between the stem-anchor and domain II leads first to hemifusion and then (f) to formation of a lipidic fusion pore.

Mechanism of membrane fusion
  • The structure described here, combined with previous knowledge, allows us to propose the following mechanism for how conformational changes in the flavivirus E protein promote membrane fusion.
  1. E associates with a CS R, through domain III probably.there is evidence for glycan-mediated interactions as well. Receptor binding leads to endosomal uptake.
  2. Reduced pH in endosome causes E dimers on virion surface to dissociate, exposing their fusion loops and letting domains I and II to flex relative to one another. Mutations alter pH threshold of fusion. Orientation for pre and postfusion structures are different. This indicates a pH-dependent hinge at domain I-domain II interface. Release of constrains by dimer contacts may let stem extend away from mebrane. Domain II turns outwards, away from virion surface, to insert its fusion loop into target cell membrane.
  3. Domain II outwardly projects. This destroys tight packing interactions on virion outer surface, allowing lateral rearrangement of E monomers. Target membranes probably catalyse trimerisation, leading to a prefusion intermediate. in which the trimer bridges host-cell and viral membranes. Its fusion loops bind  the former and TM tail anchors in the latter. 
  4. Trimer contacts form from fusion loops at trimer tip to domain I at the base. Domain II shifts and rotates, folding C terminus of sE back towards fusion loop.  Free energy released by this refolding can drive the two membranes to bend towards each other. Fusion loop insertion induces positive bilayer curvature. This may stabilise lateral surfaces of protrusions. 
  5. A hemifusion stalk forms. Its proximal leaflets are fused and distal leaflets are unfused. It is thought to be an essential intermediate.  Hemifusion stalks can ‘flicker’ open into narrow fusion pores.  Migration of the transmembrane segments along a transient pore will prevent its closing.  If TM segments of adj stem regions snap into place round tips of domains II, formation of symmettrical final structure could drive transition from stalk to pore. 
  6. Trimer reaches conformation, with stems docked along surface of domains II. Fusion loops and TM anchors are next to each other in the fused membrane.
Comparison with class I fusion
  • Class I and II have some common features.
  • During fusion transitions, the protein folds back so its 2 membrane attachment points come together in postfusion structure.
  • Class I proteins fold back by zippering up an outer layer around a central trimeric coiled coil.
  • For trimeric dengue sE, class II proteins fold back by nucleating trimer formation around an elongated fingerlike fusion domain, rearranging 2 other domains, and probably zippering an extended C-terminal stem along trimer surface.
  • Class II viral fusion proteins form trimers from monomers.
  • Class I proteins are trimeric in prefusion state.
  • In influenza HA and trimerisation of dengue E, the important trimer interactions in final state form during transition
  •  postulated prefusion intermediate is, both for class I fusion proteins and now for class II, a structure in which these central trimer contacts have formed but the zippering-up of the outer layer has not yet begun

Thursday, 24 May 2012

William Hazlitt: the First Modern Man by Duncan Wu

One of the few modern works I actually read. This biography describes the life of Romantic essayist and journalist, William Hazlitt. I first encountered his name in some Penguin book showing essays by people such as Dr Johnson. I believe it was opinions of thinkers on Shakespeare through the ages. That was years ago and I saw Hazlitt's name there, with a clear, interesting piece. I didn't read through, as I was less mature then.

Hazlitt was the son of a Unitarian minister, which meant they were radical. If you weren't an Anglican in early 19th century England, well, you were rather unfortunate. You couldn't attend Oxford and Cambridge and weren't entitled to certain privileges. The Unitarians, however were noted for being progressive and educating their daughters. A number of prosperous and liberal merchants were Unitarians and so were thinkers. Well, young William was a quick learner, and his father put a lot of pressure on him to excel, resulting in his illness. In his teens he was sent to a school meant for clever Unitarians to train them into ministers (clergymen required a good education). But the school was so progressive (it urged you to question everything, and Unitarians do not believe in a trinity) William ended up an agnostic. I rather like that. His father was disappointed as he hoped that his clever son would become a minister. Anyway somewhere in his late teens, William met a new minister in the district, a Samuel Taylor Coleridge (the poet) whom he really looked up to. At this time Coleridge was a genuis and had not collapsed to a drug-addled stupor. He had a great many ideas, far more than Wordsworth, and it was his philosophy that Wordsworth used in his poetry.  So those of you who condemn Rime of the Ancient Mariner as a melodramatic narrative and praise Wordsworth's dull pastorals as genius, recall the meanings in Coleridge, and above all, observe the power and thrill that poem sends through you - something Wordsworth never mastered. Wordsworth at his best is stately and grand, but he is not thrilling. Hazlitt used to visit Coleridge to speak to him at the height of his idolatry. It seems in his youth Hazlitt was also introduced to Wordsworth, but when he argued against Wordsworth's thinking the latter was annoyed, and even condemned Hazlitt in a poem about the latter's over-reasoning. It shows us a less benevolent side of the poet.

Eventually Hazlitt starts off as a literary critic and journalist in London, and befriends varied literary people such as Charles Lamb of Tales of Shakespeare fame, Leigh Hunt, editor of the Examiner, a radical paper, and John Hamilton Reynolds. I can't do much justice to him, but Hazlitt was a fervent radical who supported the French Revolution and revered Napoleon. For those of you accustomed to see the guy as a butcherer, let it be known that Napoleon spearheaded the aristocracy, bringing more democracy to France. Much as I deplore his intent on war, the man did do a great deal of good. Unfortunately when people like Wordsworth and Coleridge stopped supporting the Revolution Hazlitt denounced them in his articles, which led them to break up their former friendship (which wasn't much anyway). It was this fervent revolutionary instinct that led to his loss of friends.

But it was Hazlitt who wrote intelligent literary and theatrical pieces in the papers he worked for. He wrote a book on characters in Shakespeare which sold well, and for a while, was a respected critic. In the evenings, to supplement his income he gave lectures which proved to be popular.

But their is always downfall in Hazlitt's life. His acidness made him unpopular, and other critics denounced him in their articles, saying he belonged to the cockney school led by Leigh Hunt (these were not classically-educated men who had literary pretensions). It is ironic to reflect that Wordsworth, Southey and Coleridge, who eventually conformed to the establishment, are now well-known, and were respected in the Victorian era. Keats, who Hazlitt praised, only became a major figure in the late 19th century, many years after the Lake Poets gained recognition. We see in here certain aspects of Wordsworth - his egotism (he hated being criticised and would break up associations based on that), and more happily, the goodness of Charles Lamb. Hazlitt used to go over to Lamb's house and drink and talk merrily, and his wife was friends with the Lambs as well. After a certain downturn they stopped seeing each other, but when a great deal of criticism was piled on Hazlitt, Lamb retorted that despite his faults, he still admired and saw him as a friend, and believed Hazlitt to be the wisest man he knew. His conversation was better than anybody else's.

What really brought him down was the publication of Liber Amoris, an account of his affair with his landlord's daughter, who seems to have been a nymphomaniac. People thought him vulgar, and after that his books would not sell. It was this obsession with this girl that made him divorce his wife (in Scotland, but the divorce would not be recognised in England) so he could marry her. She ended up betraying him for another man.

Hazlitt eventually died in poverty and by then most of his friends had left him. I can't help feeling sorry for the man, despite his sharp remarks: he was ruled by idealism all his life. I have more sympathy for the grumpy anti-hero of exalted ideals than the likeable, popular man with no strong feelings, as Jane Eyre would agree. What we have inherited from him is modern journalism - a more personal touch - and literary criticism. While many accused Hazlitt's Shakespearean venture as not academic, it is readable, even by today's standards, and when you consider the hard language of that time, it would be much easier to read compared to today's academic texts. And profounder too than today's short amateur reviews on literature. His judgements on Hamlet are acute. We must also praise Hazlitt for remarking that his favourite actor, Edmund Kean, while brilliant at Shakespearean characters, could no bring Hamlet to life, because Hamlet's character is essentially the sort you find in a book. No one could portray his essence on stage, not even the best actor. And this is very true to this day.

Poem on Tess of the d'Urbervilles, Part 2

This is the continuation of my poem. Yes, I wrote it myself, though it is based on Hardy's novel. I have striven to adopt Victorian mannerisms in my writing, which is why it may sound old-fashioned. This is based on Chapter 2. If you're confused, Tess lives in a village called Marlott, which is I believe in a district called Blakemoor, which is in Dorset. If you don't know, just check the book.

Three Girls in White by Kate Greenaway
v.
On Blackmoor's vale the sun shone bright,
Over the pastures green and wide,
Young damsels march/flock round, side by side,
Young maidens gather garbed in white;
It is May-day, the spring is come,
Where soil sprouts/shoots grass, not arable,
(Like sublime scenes from Constable),
And nightingales begin to hum.
They tread light-footed in the spring, / Soft, soft - some part their lips to sing,
Each with a basket in her hand
With flowers white, and willow wand,
And dance like fairies in a ring.
May Dance by Kate Greenaway
vi.
They light a fire with their twigs,
Amidst the flames they start to chant,
That flicker as they sing and prance:
But then John rides by in a gig:
"I've got a noble vault!" he cries.
Shrill titters burst from our youth,
One maiden stands - silent, aloof,
With fire in her changing/melting/smouldered eyes./ Fire in many-shaded eyes.
"Tess Durbeyfield, it's thy father!"
"Don't joke about him!" she retorts,
Her cheeks glow red, her face is hot/ Her cheeks glow crimson, flushing hot -
They see her wrath and cease their blather.


She seems to come from lands afar

All nature's pride wells in revolt,
She turns her gaze to realms/sights/lands afar.

May Day by Kate Greenaway
vii.
The heads of hair gleam in the blaze:
Some golden threads, some ringlets brown,
Some noses fair, some pretty crowns,
But none is fair in every way.
No more fair is Tess's form,
Save for her innocent dark eyes,
Where haunting sorrow seems to rise;
With rose-blown cheeks, and rounded arm.
Her mouth is like a peony wild
Still wrapt in bud, and yet to bloom,
As if ashroud in Nature's womb:
Ripe years have not plucked out/unplucked the child.
viii.
Into the flames their herbs they cast,
Fragrant fennel, yellow rue,
Columbine and daisy too:
The violets taken up at last.
As if a silent prophecy
Had wove around their May-day charms.
The air is soft, the dew is balm,
Smoke towers up into the sky.
Three brothers stroll along the lane:
One a curate, one a student,
The third is of uncertain bent,
And longing looks at their terrain/ sets his gaze on their terrain./ He gazes at the grassy plain.
ix.
He longs to/yearns a dance, but it is late,
His brothers urge him to depart.
Being a man of fits and starts,
He says, "Go on. You need not wait.
I won't be long, there will be time
To curl up with your scholar's feast
And counterblast the atheists."
Over the wooden fence he climbs.


Author's note: This is not meant to be anti-atheist. I am in fact an atheist. The character of the curate is such that he wants to read a Counterblast to Agnosticism at home.

Illustrations are by Kate Greenaway, a prominent Victorian illustrator. She illustrated Robert Browning's Pied Piper of Hamelin, so the Victorians were actually more accepting of women's careers more than we like to think.

I waited long and hard for thee/you
Not doubting once thy /your perfidy. 
Now this would be interesting lines, though I've no idea how to start from there.