A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs.

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.

Strategies for rapid production of crystallization quality coatomer WD40 domains.

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 ß'-COPI subunits to bind these membrane protein "clients" for ER retrieval. These "αWD40" and "ß'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 ß'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.

CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3 determined using a bivalent Fab as fiducial marker.

Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5 Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100 kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab-LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins.

An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking.

ß-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

The Structural Biology of Eastern Equine Encephalitis Virus, an Emerging Viral Threat.

Alphaviruses are arboviruses that cause arthritis and encephalitis in humans. Eastern Equine Encephalitis Virus (EEEV) is a mosquito-transmitted alphavirus that is implicated in severe encephalitis in humans with high mortality. However, limited insights are available into the fundamental biology of EEEV and residue-level details of its interactions with host proteins. In recent years, outbreaks of EEEV have been reported mainly in the United States, raising concerns about public safety. This review article summarizes recent advances in the structural biology of EEEV based mainly on single-particle cryogenic electron microscopy (cryoEM) structures. Together with functional analyses of EEEV and related alphaviruses, these structural investigations provide clues to how EEEV interacts with host proteins, which may open avenues for the development of therapeutics.

Structural and biochemical insights into flavivirus proteins.

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.

A novel chloroplast super-complex consisting of the ATP synthase and photosystem I reaction center.

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.

Cryo-EM structure of eastern equine encephalitis virus in complex with heparan sulfate analogues.

Eastern equine encephalitis virus (EEEV), a mosquito-borne icosahedral alphavirus found mainly in North America, causes human and equine neurotropic infections. EEEV neurovirulence is influenced by the interaction of the viral envelope protein E2 with heparan sulfate (HS) proteoglycans from the host's plasma membrane during virus entry. Here, we present a 5.8-Å cryoelectron microscopy (cryo-EM) structure of EEEV complexed with the HS analog heparin. "Peripheral" HS binding sites were found to be associated with the base of each of the E2 glycoproteins that form the 60 quasi-threefold spikes (q3) and the 20 sites associated with the icosahedral threefold axes (i3). In addition, there is one HS site at the vertex of each q3 and i3 spike (the "axial" sites). Both the axial and peripheral sites are surrounded by basic residues, suggesting an electrostatic mechanism for HS binding. These residues are highly conserved among EEEV strains, and therefore a change in these residues might be linked to EEEV neurovirulence.

A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV).

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Šresolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization.

Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design.

Structural biology of Zika virus and other flaviviruses.

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.

Structural studies of Chikungunya virus maturation.

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.

A human antibody against Zika virus crosslinks the E protein to prevent infection.

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.

Pathways of Transmembrane Electron Transfer in Cytochrome bc Complexes: Dielectric Heterogeneity and Interheme Coulombic Interactions.

The intramembrane cytochrome bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus and the cytochrome b6f complex, which functions in oxygenic photosynthesis, utilize two pairs of b-hemes in a symmetric dimer to accomplish proton-coupled electron transfer. The transmembrane electron transfer pathway in each complex was identified through the novel use of heme Soret band excitonic circular dichroism (CD) spectra, for which the responsible heme-heme interactions were determined from crystal structures. Kinetics of heme reduction and CD amplitude change were measured simultaneously. For bc1, in which the redox potentials of the transmembrane heme pair are separated by 160 mV, heme reduction occurs preferentially to the higher-potential intermonomer heme pair on the electronegative (n) side of the complex. This contrasts with the b6f complex, where the redox potential difference between transmembrane intramonomer p- and n-side hemes is substantially smaller and the n-p pair is preferentially reduced. Limits on the dielectric constant between intramonomer hemes were calculated from the interheme distance and the redox potential difference, ΔEm. The difference in preferred reduction pathway is a consequence of the larger ΔEm between n- and p-side hemes in bc1, which favors the reduction of n-side hemes and cannot be offset by decreased repulsive Coulombic interactions between intramonomer hemes.

Trans-membrane Signaling in Photosynthetic State Transitions: REDOX- AND STRUCTURE-DEPENDENT INTERACTION IN VITRO BETWEEN STT7 KINASE AND THE CYTOCHROME b6f COMPLEX.

Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.

Role of domain swapping in the hetero-oligomeric cytochrome b6f lipoprotein complex.

Domain swapping that contributes to the stability of biologically crucial multisubunit complexes has been implicated in protein oligomerization. In the case of membrane protein assemblies, domain swapping of the iron-sulfur protein (ISP) subunit occurs in the hetero-oligomeric cytochrome b6f and bc1 complexes, which are organized as symmetric dimers that generate the transmembrane proton electrochemical gradient utilized for ATP synthesis. In these complexes, the ISP C-terminal predominantly ß-sheet extrinsic domain containing the redox-active [2Fe-2S] cluster resides on the electrochemically positive side of each monomer in the dimeric complex. This domain is bound to the membrane sector of the complex through an N-terminal transmembrane α-helix that is "swapped' to the other monomer of the complex where it spans the complex and the membrane. Detailed analysis of the function and structure of the b6f complex isolated from the cyanobacterium Fremyella diplosiphon SF33 shows that the domain-swapped ISP structure is necessary for function but is not necessarily essential for maintenance of the dimeric structure of the complex. On the basis of crystal structures of the cytochrome complex, the stability of the cytochrome dimer is attributed to specific intermonomer protein-protein and protein-lipid hydrophobic interactions. The geometry of the domain-swapped ISP structure is proposed to be a consequence of the requirement that the anchoring helix of the ISP not perturb the heme organization or quinone channel in the conserved core of each monomer.

Traffic within the cytochrome b6f lipoprotein complex: gating of the quinone portal.

The cytochrome bc complexes b6f and bc1 catalyze proton-coupled quinol/quinone redox reactions to generate a transmembrane proton electrochemical gradient. Quinol oxidation on the electrochemically positive (p) interface of the complex occurs at the end of a narrow quinol/quinone entry/exit Qp portal, 11 Å long in bc complexes. Superoxide, which has multiple signaling functions, is a by-product of the p-side quinol oxidation. Although the transmembrane core and the chemistry of quinone redox reactions are conserved in bc complexes, the rate of superoxide generation is an order of magnitude greater in the b6f complex, implying that functionally significant differences in structure exist between the b6f and bc1 complexes on the p-side. A unique structure feature of the b6f p-side quinol oxidation site is the presence of a single chlorophyll-a molecule whose function is unrelated to light harvesting. This study describes a cocrystal structure of the cytochrome b6f complex with the quinol analog stigmatellin, which partitions in the Qp portal of the bc1 complex, but not effectively in b6f. It is inferred that the Qp portal is partially occluded in the b6f complex relative to bc1. Based on a discrete molecular-dynamics analysis, occlusion of the Qp portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Qp portal and is inferred to be a cause of enhanced superoxide production. This study attributes a novel (to our knowledge), structure-linked function to the otherwise enigmatic chlorophyll-a in the b6f complex, which may also be relevant to intracellular redox signaling.

Structure-function of cyanobacterial outer-membrane protein, Slr1270: homolog of Escherichia coli drug export/colicin import protein, TolC.

Compared to thylakoid and inner membrane proteins in cyanobacteria, no structure-function information is available presently for integral outer-membrane proteins (OMPs). The Slr1270 protein from the cyanobacterium Synechocystis 6803, over-expressed in Escherichia coli, was refolded, and characterized for molecular size, secondary structure, and ion-channel function. Refolded Slr1270 displays a single band in native-electrophoresis, has an α-helical content of 50-60%, as in E. coli TolC with which it has significant secondary-structure similarity, and an ion-channel function with a single-channel conductance of 80-200pS, and a monovalent ion (K(+):Cl(-)) selectivity of 4.7:1. The pH-dependence of channel conductance implies a role for carboxylate residues in channel gating, analogous to that in TolC.

Internal lipid architecture of the hetero-oligomeric cytochrome b6f complex.

The role of lipids in the assembly, structure, and function of hetero-oligomeric membrane protein complexes is poorly understood. The dimeric photosynthetic cytochrome b6f complex, a 16-mer of eight distinct subunits and 26 transmembrane helices, catalyzes transmembrane proton-coupled electron transfer for energy storage. Using a 2.5 Å crystal structure of the dimeric complex, we identified 23 distinct lipid-binding sites per monomer. Annular lipids are proposed to provide a connection for super-complex formation with the photosystem-I reaction center and the LHCII kinase enzyme for transmembrane signaling. Internal lipids mediate crosslinking to stabilize the domain-swapped iron-sulfur protein subunit, dielectric heterogeneity within intermonomer and intramonomer electron transfer pathways, and dimer stabilization through lipid-mediated intermonomer interactions. This study provides a complete structure analysis of lipid-mediated functions in a multi-subunit membrane protein complex and reveals lipid sites at positions essential for assembly and function.

A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.