Zika virus (ZIKV) is an enveloped, icosahedral flavivirus that has structural and functional similarities to other human flavivirus pathogens such as dengue (DENV), West Nile (WNV) and Japanese ...encephalitis (JEV) viruses. ZIKV infections have been linked to fetal microcephaly and the paralytic Guillain-Barré syndrome. This review provides a comparative structural analysis of the assembly, maturation and host-cell entry of ZIKV with other flaviviruses, especially DENV. We also discuss the mechanisms of neutralization by antibodies.
The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The ...cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and β′-COPI subunits to bind these membrane protein “clients” for ER retrieval. These “αWD40” and “β'WD40” domains are structural homologs that demonstrate distinct selectivity for client proteins. However, elucidation of the atomic-level principles of coatomer-client interactions has been challenging due to the tendency of αWD40 domain to undergo aggregation during expression and purification. Here we describe a rapid recombinant production strategy from E. coli, which substantially enhances the quality of the purified αWD40 domain. The αWD40 purification and crystallization are completed within one day, which minimizes aggregation losses and yields a 1.9 Å resolution crystal structure. We demonstrate the versatility of this strategy by applying it to purify the β′WD40 domain, which yields crystal structures in the 1.2–1.3 Å resolution range. As an alternate recombinant production system, we develop a cost-effective strategy for αWD40 production in human Expi293 cells. Finally, we suggest a roadmap to simplify these protocols further, which is of significance for the production of WD40 mutants prone to rapid aggregation. The WD40 production strategies presented here are likely to have broad applications because the WD40 domain represents one of the largest families of biomolecular interaction modules in the eukaryotic proteome and is critical for trafficking of host as well as viral proteins such as the SARS-CoV-2 spike protein.
•N-terminal WD40 domains of α- and β′COPI bind dibasic motifs in client protein tail.•α- and β′WD40 domain binding counters client protein leakage during secretion.•αWD40 and β′WD40 production is described in E. coli and mammalian systems.•Rational production schemes suggested for challenging αWD40 and β′WD40 mutants.•Applications in atomic-level investigations of secretory metabolism and disease.
Structural studies of Chikungunya virus maturation Yap, Moh Lan; Klose, Thomas; Urakami, Akane ...
Proceedings of the National Academy of Sciences - PNAS,
12/2017, Volume:
114, Issue:
52
Journal Article
Peer reviewed
Open access
Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. ...Here we present a cryo-EM, 6.8-Å resolution structure of an “immature” Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protecting a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.
Aspects of the crystal structures of the hetero-oligomeric cytochrome
bc
1 and
b
6
f (“bc”) complexes relevant to their electron/proton transfer function and the associated redox reactions of the ...lipophilic quinones are discussed. Differences between the
b
6
f and
bc
1 complexes are emphasized. The cytochrome
bc
1 and
b
6
f dimeric complexes diverge in structure from a core of subunits that coordinate redox groups consisting of two
bis-histidine coordinated hemes, a heme
b
n and
b
p on the electrochemically negative (n) and positive (p) sides of the complex, the high potential 2Fe–2S cluster and
c-type heme at the p-side aqueous interface and aqueous phase, respectively, and quinone/quinol binding sites on the n- and p-sides of the complex. The
bc
1 and
b
6
f complexes diverge in subunit composition and structure away from this core.
b
6
f Also contains additional prosthetic groups including a
c-type heme
c
n on the n-side, and a chlorophyll
a and β-carotene.
Common structure aspects; functions of the symmetric dimer
. (I)
Quinone exchange with the bilayer. An inter-monomer protein-free cavity of approximately 30
Å along the membrane normal
×
25
Å (central inter-monomer distance)
×
15
Å (depth in the center), is common to both
bc
1 and
b
6
f complexes, providing a niche in which the lipophilic quinone/quinol (Q/QH
2) can be exchanged with the membrane bilayer. (II)
Electron transfer. The dimeric structure and the proximity of the two hemes
b
p on the electrochemically positive side of the complex in the two monomer units allow the possibility of two alternate routes of electron transfer across the complex from heme
b
p to
b
n: intra-monomer and inter-monomer involving electron cross-over between the two hemes
b
p. A structure-based summary of inter-heme distances in seven
bc complexes, representing mitochondrial, chromatophore, cyanobacterial, and algal sources, indicates that, based on the distance parameter, the intra-monomer pathway would be favored kinetically. (III)
Separation of quinone binding sites. A consequence of the dimer structure and the position of the Q/QH
2 binding sites is that the p-side QH
2 oxidation and n-side Q reduction sites are each well separated. Therefore, in the event of an overlap in residence time by QH
2 or Q molecules at the two oxidation or reduction sites, their spatial separation would result in minimal steric interference between extended Q or QH
2 isoprenoid chains. (IV)
Trans-membrane QH
2
/Q transfer. (i) n/p-side QH
2/Q transfer may be hindered by lipid acyl chains; (ii) the shorter less hindered inter-monomer pathway across the complex would not pass through the center of the cavity, as inferred from the n-side antimycin site on one monomer and the p-side stigmatellin site on the other residing on the same surface of the complex. (V)
Narrow p-side portal for QH
2
/Q passage. The 2Fe–2S cluster that serves as oxidant, and whose histidine ligand serves as a H
+ acceptor in the oxidation of QH
2, is connected to the inter-monomer cavity by a narrow extended portal, which is also occupied in the
b
6
f complex by the 20 carbon phytyl chain of the bound chlorophyll.
► Comparative discussion of crystal structures, biochemical properties, and functions of cytochrome
bc
1 complexes and the
b
6
f complex in oxygenic photosynthesis. ► Structure-function of lipids integral to the complex. ► Differences in Q cycle in
bc
1 and
b
6
f complexes. ► Steric problems in entry/exit of lipophilic quinol/quinone in
p-side portal of cytochrome
bc complexes. ► Implications of structure for intra- and inter-monomer pathways of electron transfer.
Considering information in the crystal structures of the cytochrome b(6)f complex relevant to the rate-limiting step in oxygenic photosynthesis, it is enigmatic that electron transport in the complex ...is not limited by the large distance, approximately 26 Å, between the iron-sulfur cluster (ISP) and its electron acceptor, cytochrome f. This enigma has been explained for the respiratory bc(1) complex by a crystal structure with a greatly shortened cluster-heme c(1) distance, leading to a concept of ISP dynamics in which the ISP soluble domain undergoes a translation-rotation conformation change and oscillates between positions relatively close to the cyt c(1) heme and a membrane-proximal position close to the ubiquinol electron-proton donor. Comparison of cytochrome b(6)f structures shows a variation in cytochrome f heme position that suggests the possibility of flexibility and motion of the extended cytochrome f structure that could entail a transient decrease in cluster-heme f distance. The dependence of cyt f turnover on lumen viscosity is consistent with a role of ISP - cyt f dynamics in determination of the rate-limiting step under conditions of low light intensity. Under conditions of low light intensity and proton electrochemical gradient present, for example, under a leaf canopy, it is proposed that a rate limitation of electron transport in the b(6)f complex may also arise from steric constraints in the entry/exit portal for passage of the plastoquinol and -quinone to/from its oxidation site proximal to the iron-sulfur cluster.
The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. ...Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117. The antibody had been shown to prevent fetal infection and demise in mice. The structure shows that ZIKV-117 Fabs cross-link the monomers within the surface E glycoprotein dimers as well as between neighbouring dimers, thus preventing the reorganization of E protein monomers into fusogenic trimers in the acidic environment of endosomes.
As much as two-thirds of the proton gradient used for transmembrane free energy storage in oxygenic photosynthesis is generated by the cytochrome b₆f complex. The proton uptake pathway from the ...electrochemically negative (n) aqueous phase to the n-side quinone binding site of the complex, and a probable route for proton exit to the positive phase resulting from quinol oxidation, are defined in a 2.70-Å crystal structure and in structures with quinone analog inhibitors at 3.07 Å (tridecyl-stigmatellin) and 3.25-Å (2-nonyl-4-hydroxyquinoline N-oxide) resolution. The simplest n-side proton pathway extends from the aqueous phase via Asp20 and Arg207 (cytochrome b₆ subunit) to quinone bound axially to heme cn. On the positive side, the heme-proximal Glu78 (subunit IV), which accepts protons from plastosemiquinone, defines a route for H⁺ transfer to the aqueous phase. These pathways provide a structure-based description of the quinone-mediated proton transfer responsible for generation of the transmembrane electrochemical potential gradient in oxygenic photosynthesis.
Several 'super-complexes' of individual hetero-oligomeric membrane protein complexes, whose function is to facilitate intra-membrane electron and proton transfer and harvesting of light energy, have ...been previously characterized in the mitochondrial cristae and chloroplast thylakoid membranes. We report the presence of an intra-membrane super-complex dominated by the ATP-synthase, photosystem I (PSI) reaction-center complex and the ferredoxin-NADP+ Reductase (FNR) in the thylakoid membrane. The presence of the super-complex has been documented by mass spectrometry, clear-native PAGE and Western Blot analyses. This is the first documented presence of ATP synthase in a super-complex with the PSI reaction-center located in the non-appressed stromal domain of the thylakoid membrane.
•Flaviviruses are fastest spreading arthropod borne viruses causing fever to death.•Atomic details of viral proteins are crucial to understand fundamental biology.•Summary of major milestones in ...flavivirus structural biology in last 25 years.•Analysis of protein biochemistry in established and emerging flaviviruses.•Prediction of antibody candidates for neutralization of emerging flaviviruses.
Flaviviruses are the fastest spreading arthropod-borne viruses that cause severe symptoms such as hepatitis, hemorrhagic fever, encephalitis, and congenital deformities. Nearly 40 % of the entire human population is at risk of flavivirus epidemics. Yet, effective vaccination is restricted only to a few flaviviruses such as yellow fever and Japanese encephalitis viruses, and most recently for select cases of dengue virus infections. Despite the global spread of dengue virus, and emergence of new threats such as Zika virus and a new genotype of Japanese encephalitis virus, insights into flavivirus targets for potentially broad-spectrum vaccination are limited. In this review article, we highlight biochemical and structural differences in flavivirus proteins critical for virus assembly and host interactions. A comparative sequence analysis of pH-responsive properties of viral structural proteins identifies trends in conservation of complementary acidic-basic character between interacting viral structural proteins. This is highly relevant to the understanding of pH-sensitive differences in virus assembly in organelles such as neutral ER and acidic Golgi. Surface residues in viral interfaces identified by structural approaches are shown to demonstrate partial conservation, further reinforcing virus-specificity in assembly and interactions with host proteins. A comparative analysis of epitope conservation in emerging flaviviruses identifies therapeutic antibody candidates that have potential as broad spectrum anti-virals, thus providing a path towards development of vaccines.
The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance ...and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.
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