abstract - biorxiv.org 20, 2018 · abstract membrane fusion ... j c miner , p w fenimore , w...

Abstract

Membrane fusion proteins are responsible for viral entry into host cells– a crucial first step in viralinfection. These proteins undergo large conformational changes from pre-fusion to fusion initiation struc-tures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged astrimers. Structural information for both pre-fusion and fusion initiation states is critical for understandingvirus neutralization by the host immune system. In the case of Ebola glycoprotein (GP) and Zika envelopeprotein (Zika E), pre-fusion state structures have been identified experimentally, but only partial structuresof fusion initiation states have been described. While the fusion initiation structure is in an energeticallyunfavorable state that is difficult to solve experimentally, the existing structural information combinedwith computational approaches enabled the modeling of fusion initiation state structures of both proteins.These structural models provide an improved understanding of four different neutralizing antibodies inthe prevention of viral host entry.

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not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was. http://dx.doi.org/10.1101/285130doi: bioRxiv preprint first posted online Mar. 20, 2018;

http://dx.doi.org/10.1101/285130

Structural transition and antibody binding of Ebola GP andZika E proteins from pre-fusion to fusion-initiation states

A Lappala-Vernon1, W Nishima1,2, J C Miner1, P W Fenimore1, W Fischer1, P THraber1, M Zhang3, B H McMahon1, C-S Tung1∗

1Los Alamos National Laboratory, Los Alamos, 87545 New Mexico 2New MexicoConsortium, Los Alamos, 87545 New Mexico

3University of Georgia, Athens, 30602 Georgia

March 19, 2018

IntroductionEnveloped viruses employ a common mechanism to enter the host cell (1). The first steps, receptor bind-ing and membrane fusion, are initiated by the envelope protein (2–4). While specific details vary amongdifferent viruses, the envelope proteins invariably go through a large conformational change (5, 6) beforeinitiating membrane fusion. These large conformational changes allow the envelope protein to assume anextended fusion initiation conformation: the envelope protein in the fusion initiation state is able to bridgeacross the viral- and the host membranes, subsequently bringing the two membranes in close proximity andstarting the fusion process (7, 8). Viral neutralization by antibodies may involve binding to the fusion-statestructure or inhibiting its formation. Therefore, viral envelope proteins are important foci for developmentof vaccines and therapeutics. Recent intense research focus on Ebola and Zika viruses has provided newdata for structural modeling of these transitions.

Structural data for a number of viral envelope proteins are available in the Protein Data Bank (PDB) (9).Many of these known structures correspond to envelope proteins in the pre-fusion state, and some of thefusion-state structures only correspond to a partial molecule (usually in a low pH environment). To date,there are no structures for complete viral envelope proteins in the fusion initiation state; understandingthe mechanics of the conformational change from pre-fusion to the fusion initiation state requires such adescription.

Directly determining fusion-state structures for complete viral envelope proteins by experimental meth-ods is difficult; molecular modeling offers a readily applicable alternative means to structural characteriza-tion. We describe the use of a knowledge-based methodology (homology modeling) to develop structuresof viral envelope proteins in the fusion initiation state. We further extend the basic idea of homology mod-eling to include a simple concept “proteins and protein domains that fold similarly interact similarly”, asa result, developing structural models of envelope protein-antibody complexes. In this work we focus onenvelope proteins from Ebola and Zika viruses. Ebolavirus causes Ebola hemorrhagic fever, a severe andhighly lethal infection: the 2013-2015 West African Ebolavirus epidemic (December 2013 – 2015) resultedin approximately 11,000 confirmed deaths and 28,000 suspected cases (10). Zika virus, in contrast, causes abrief, relatively mild illness, but has been linked to congenital microcephaly and Guillan-Barre Syndrome inhumans (11, 12); in mouse models, Zika virus causes microcephaly (13), as well as damage to the male re-productive system (14) and to adult neural stem cells (15). At the time of publication, eighty-four countries,territories and subnational areas report Zika transmission (16). Ebola and Zika viruses represent persistentthreats to public health; there are limited options available for treatment or prevention of either virus. Al-though they are phylogenetically distinct viruses, their fusion subunits both adopt a trimer structure: in thisstudy, we investigate some of the similarities, differences, and consequences of these fusion-state structures.We present the following models for Ebola GP and Zika E proteins:

1. A trimer model of Ebola GP in fusion initiation state with NPC1 receptor and neutralizing antibodies.

2. A trimer model of Zika E in the fusion initiation state with neutralizing antibodies and the surrounding9-mer structure of Zika E protein in the pre-fusion state with neutralizing antibodies.

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Our modeling approach is general and comprehensive, and can be used for developing structures of otherpathogen proteins in their functional states for understanding their functions; the developed structure-basedknowledge can further add to sequence-based information and improve vaccine design for viruses and otherpathogens. Importantly, all of the developed models are testable experimentally, potentially leading to thediscovery new targets for drugs and vaccines, and optimization of known vaccine- and drug targets.

Ebola Envelope Protein GP1/GP2 and Zika Envelope Protein EEbola has a small (∼18-19 kb) negative-stranded RNA genome (17) that encodes eight viral proteins. Enve-lope glycoprotein (GP) is the viral surface protein responsible for host cell entry (18, 19) and has a sequenceof 676 residues in all known Ebola strains. The N-terminus (residues 1-32) forms a signal peptide and theremaining protein residues (33-676) are collectively referred to as the GP portion. The host endoproteasefurin cleaves GP into two segments: GP1 (residues 33-501) and GP2 (residues 502-676) (20). GP1 is re-sponsible for receptor binding to the host cell (21, 22) while GP2 acts as a class I viral fusion protein (23).Several functional domains of Ebola GP are denoted in Fig. 1. Like Influenza and HIV envelope proteins,Ebola GP is a homotrimer in its functional state (24).

Since the 2015 epidemic, significantly more Ebolavirus sequence information has become available,including sequences of GP (more than 1,000 unique Ebola GP sequences can be found in the current NCBIEbolavirus Database) (25). Significant efforts have been devoted towards solving the GP structure usingvarious experimental approaches including x-ray crystallography (24, 26–28), NMR (29, 30) and ElectronMicroscopy (31, 32). Through this work, domain structures have been solved in different functional states,as has the trimer structure of the Ebola GP mucin-like region deletion mutant (GPmuc) in the pre-fusionstate (24). All of these pieces of information contribute to a basis for developing a structural model of theGP in the fusion initiation state.

Zika virus is a member of the virus family Flaviviridae (33) that includes Dengue and West Nile viruses;it possesses a non-segmented, single-stranded, positive-sense RNA genome (10 kb) (34). The urgency infinding ways to combat the virus has increased significantly since a causal relation between Zika and apop-tosis of human neurons was discovered (11). For EBOV, the envelope protein (GP) forms homotrimers thatare distributed sparsely on the surface of the virus, as in influenza and HIV. By contrast, Zika Es form ho-modimers and cover the entire viral surface (the capsid comprises 90 homodimers, arranged as 30 rhombicfaces with 3 dimers each) (35, 36). However, the partial structure of the Zika E in the fusion initiation state(at low pH) shows a trimer arrangement. Determining how Zika E transforms from a pre-fusion dimer to afusion-state trimer presents a considerable challenge to the structural modeling community.

Methods and Materials

Homology modeling using a Motif-Matching Fragment Assembly methodMotif-Matching Fragment Assembly Method (MMFA) was used for complex homology modeling (de-scribed in our earlier work (37)). This approach was used to develop structural models of neutralizingantibodies binding to its targeted fusion proteins in their functional unit and state.

Structure alignment and superpositionTo develop fusion initiation state structure of the GP protein using the spring-loaded mechanism, domainstructural assembly was accomplished by superposition of structurally matched regions (e.g., helical seg-ments). The MatchMaking option in Structural Comparison Tool of Chimera (38) is used for structurealignment and superposition of the structurally matched segments.

Structural refinementMolecular dynamics simulations of Zika and Ebola models were performed in a standard manner as de-scribed in Hess et al. (39). Initially, structures were energy-minimized with a steepest descent minimizationalgorithm, after which the protein underwent a 100 ps NVT (constant number of particles, volume andtemperature) equilibration step, followed by NPT (constant number of particles, pressure and temperature)equilibration for another 100 ps. The production run was performed under standard NPT conditions at 300Kfor 100 ns.

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(a)

(b) (c)

(d)

Figure 1: Structures of Ebola GP corresponding to the pre-fusion and fusion initiation states. a. (Top) Atrimer structure of GP1/GP2 in the pre-fusion state and (Bottom) GP2 in the fusion initiation state weresolved using x-ray crystallography (PDB accession codes 3CSY and 2EBO, respectively). b. Model of thepre-fusion structure of the GP2 trimer with a spring-loaded mechanism. c. Hairpin loop structure (residues510 to 559) of GP2, including the fusion peptide (residues 524-539, red), remodeled into a conformationwith the fusion peptide pointing toward the host cell surface. d. GP1/GP2 trimer in the fusion initiationstate bound to the NPC1 receptor. Viral membrane is oriented at the bottom of each panel.

GraphicsMolecular graphics images were produced using the UCSF Chimera package (40) from the Resource forBio-computing, Visualization and Informatics at the University of California, San Francisco.

Results

Pre-fusion and fusion initiation state structures of EBOV GP (‘spring-loaded model’)The crystal structure of EBOV GP1/GP2 has been described in the pre-fusion state (accession code: 3CSY) (24).In this structure, GP1 and GP2 exist as a trimer, with the GP2 bound from the outside and the inside of theGP1 proteins. A partial structure of GP2 (lacking N-terminal and C-terminal domains) in the fusion initia-tion state has also been solved at low pH (accession code: 2EBO) (26). The pre-fusion and fusion initiationstates of GP2 show different folds (see Fig. 1a) with a small triple-helix structure shown in both (highlightedin magenta).

In the pre-fusion structure, the yellow/magenta regions of GP2 are wrapped around the GP1 trimer onthe outside of the GP1 trimer. For the yellow helices to stack on top of the magenta helices as indicated(PDB: 2EBO), they must rearrange from the outside to the inside of the GP1 trimer. A close inspectionof the GP structure in the pre-fusion state reveals a connection between two GP1 proteins in the trimerthrough loops S90 to P93 and P126 to R130. To accomplish the transition step, this weak bridge needsto be disconnected to allow the N-terminal part of GP2 to move from the outside of the GP1 trimer to theinside.

The C-terminal region of GP2 (highlighted in blue, Fig. 1a) needs to ‘peel off’ from the central triplehelix of the fusion initiation state in order for GP2 to bind and fit into the internal part of the GP1 trimer.Since the C-terminal end of GP2 contains the trans-membrane domain that points toward the viral surface- and is less likely to be immunogenic - our model is truncated at residue 599. To account for the largeconformational changes during the pre-fusion to fusion transition, a spring-loaded model is applied (41, 42).This same model was considered as the mechanism for the Ebola GP structural transition in a previous studyby White et al. (43), and is supported by structural information of Ebola GP in two different states (Fig. 1a).

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The spring-loaded model suggests that the two GP2 helices in the pre-fusion state structure (3CSY) shouldbe stacked together to form one long helix, as shown in the fusion initiation state structure (2EBO). Whenthe two helices become one long helix, the N-terminal region of GP2 (residues 502-555) will be stacked ontop of the long triple helix and pointed radially outward (Fig. 1b).

The known structures of viral envelope proteins in the fusion initiation state usually show a trimerarrangement in a compact state (6) where the fusion peptides are tightly localized on top of the trimer andexposed to the host cell membrane (44). The GP2 structure (Fig. 1c) has the fusion peptides (residues514-539, highlighted in red) separated and aligned outwards, parallel to the viral membrane, instead ofupward toward the host cell membrane. In order to develop a model that matches known fusion initiationstate structures, the sole β-hairpin structure in GP2 (residues 517-520, 543-546) are fitted to a trefoil foldβ-hairpin template (PDB: 2F2F) at each trimer. The modeled β-hairpins in the trefoil fold are then dockedon top of the yellow triple helix using the disulfide bond between C-511 and C-556 as a constraint. Finally,the low pH fusion loop structure (PDB: 2RLJ) is used as a template to model the fusion peptide structure(residues 521-542). The final structure of the GP2 trimer in the fusion initiation state is shown in Fig. 1c.

Niemann-Pick C1 (NPC1) has been identified as the entry receptor for EBOV GP (45), which mustbe primed to a fusion-competent state before binding an NPC1 receptor. The priming process includescleavage of the mucin-like domain, the glycan cap and the outmost strand of the proposed receptor bindingregion (24, 46). Using the NPC1-bound GP1 structure (PDB: 5F1B) as a template, primed GP1-GP2-NPC1complex in the fusion initiation state is modeled (Fig. 1d).

Based on these results we propose that the connecting bridge in the trimer (S90 to P93, P126 to R130)serves as a gate for the pre-fusion to fusion structural transition. If this gate is locked, the structural transitionstep is inhibited, which impedes viral replication.

Pre-fusion and fusion structure of the Zika EThe basic unit of the Zika E protein, based on structures derived from x-ray crystallography and cryo-EM (PDB accession codes: 5JHM, 5LBS, 3CSY), shows the protein in a head-to-tail dimer arrangement(Fig. 2a). While the structure of Zika E in the fusion initiation state is not known, the partial Zika E structureof the related Dengue virus (DENV), has been described in its fusion initiation state (PDB accession code:1OK8). Due to the high sequence similarity of DENV and Zika, it is a simple exercise to model the structureof Zika E in the fusion initiation state using the homology modeling approach (Fig. 2b).

Based on the known structure, Zika E can be divided into three domains: domain I (residues 1-52, 132-193, 280-296), domain II (residues 53-131, 193-276) and domain III (residues: 297-501). In this work, wefurther divide domain III into domain III-1 (residues 297-403) and domain III-2 (residues 404-501). DomainIII-2 is absent in the fusion initiation state structure. Converting Zika E from 2-fold to 3-fold symmetry isa significant modeling challenge. Schematic representations of the flavivirus membrane-fusion mechanismhave been proposed (47, 48), involving (i) a low-pH induced dissociation of the Zika E dimer, (ii) outwardprojection of Zika E monomer, and finally, (iii) formation of the fusion-state trimer. The proposed processdoes not provide information about the full Zika E structure in the fusion initiation state, so the relativeposition and orientation of the trimer with respect to the rest of the Zika E monomers on the surface ofthe virus requires further elucidation. Here, we propose a direct path for the Zika E pre-fusion to fusionstructural conversion.

The cryo-EM structure of Zika E (PDB: 5IRE) is shown in Fig. 2c. In this representation, three dimersform a rhombus-shaped hexamer (highlighted by the parallelogram), and thirty copies of this hexamer aresufficient to cover the entire surface of the virus. These hexamers form 3- and 5-fold symmetries at theirvertices, where the ends of either three or five Zika E proteins meet. The basic unit used to model thepre-fusion to fusion transition are three dimers that form a 3-fold symmetry. Since domain III-2 is absentin the DENV E protein fusion initiation state structure, we propose that this region serves as an anchor forthe structural transition, and domains I, II and III-1 from each of the three Zika E molecules are involved inthe structural transition. This transition is accomplished through a 90◦ rotation of domain I/II. The bindingmodes of I/II and III-1 are different between the pre-fusion (3CSY) and fusion (1OK8-derived) structures.The I/II and III-1 binding mode for 1OK8 upsets the connection between III-1 and III-2, thus the I/II/III-1arrangement of the pre-fusion structure is used in the final fusion initiation state (spike in Fig. 2d).

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spike5-fold

3-fold

(a) (b)

(c) (d)

Figure 2: The pre-fusion and fusion structures of the Zika E protein. a. The 3.8 Angstrom cryo-EMstructure of the Zika E dimer. b. Left Partial structure of DENV E, and Right modeled structure of ZikaE in their fusion initiation states. These structures are divided into four domains, highlighted as follows:(I: red, II: yellow, III-1: blue, III-2: cyan). c. The 180 copies of Zika E cover (from cryo EM) the surfaceof the viral capsid, with intersection points of 3- and 5-fold symmetries marked. d. A model of side- andtop-views of the Zika E trimer (marked as a spike) in the fusion initiation state.

Neutralizing antibody blocks viral entry by pre-fusion and fusion initiation stateinteractionsNeutralizing antibodies inhibit pathogen entry into the host cell. Due to the fact that this is a complexprocess (49), blocking viral host-cell entry may occur at multiple stages. Since the conformational transitionof the viral envelope protein from a pre-fusion to a fusion initiation state is a crucial step in host-cell entry,we explore different neutralizing antibodies, and their roles in blocking cell entry, by studying the bindingof antibodies to Zika E in pre-fusion and fusion initiation states.

Antibody KZ52 blocks EBOV entry by preventing GP structural transition from a pre-fusion to afusion initiation state

The KZ52-bound Zaire-Ebola GP trimer in the pre-fusion state has been solved using x-ray crystallog-raphy (PDB accession code: 3CSY). Residues of the GP that are responsible for KZ52 binding includeGP1(residues 42/43) and GP2(residues 505-511/513-514/549-553/556). If the N-terminal portion of GP2cannot peel off from the outside and move to the inside of the GP1 trimer then the conformational transi-tion to the fusion initiation state structure cannot be completed. Therefore, binding of KZ52 to EBOV GPprevents the structural transition of the protein to the fusion initiation state. A similar binding mechanismwas observed for the binding of 16F6 to Sudan-Ebola GP (PDB accession code: 3S88). Therefore, we mayargue that 16F6 blocks cell entry by preventing the structural transition of GP from a pre-fusion to a fusioninitiation state.

Antibody mAb100 blocks EBOV entry through two different mechanisms

Antibody mAb100, when used in conjunction with mAb114, can protect non-human primates against allsigns of Ebola virus disease (50). The crystal structure of mAb100-bound EBOV GP (PDB accession code:5FHC) shows that mAb100 binding occludes access to the cathepsin-cleavage loop Fig. 3, thus preventingviral entry. It is a straightforward exercise to develop a model of Ebola GP trimer with bound mAb100by superimposing the GP structure in 5FHC with that in 3CSY. In this model, mAb100 interacts with two

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mAb100

GP2GP2

GP1 trimer

GP1 trimer

GP2

mAb100

(a) (b)

Figure 3: The mAb100 antibody can bind to the EBOV GP trimer in both pre-fusion and fusion initiationstates. a. Modeled mAb100 binding to two GP2 monomers when the GP trimer is in the pre-fusion state,preventing the fusion initiation state transition. b. A post-transition model, where the mAb100 bindingsite on GP2 is exposed at the top of the trimer, allowing the antibody to bind. The model shows that upto three mAb100 antibodies can bind to the GP trimer in the fusion initiation state structure without stericinterference.

copies of GP2 in the trimer. As a result, mAb100 directly blocks the transition from pre-fusion to fusioninitiation state. From the perspective of 5FHC, mAb100 recognises and binds to a loop near the fusionpeptide in GP2. Using this binding mode, we develop a model of mAb100 binding to a GP trimer in thefusion initiation state. It was further shown that up to three mAb100 can bind simultaneously to a GPtrimer in the fusion initiation state. While the fusion peptide is exposed when the GP trimer is in the fusioninitiation state structure, the exposure is blocked when mAb100 binds to the viral GP.

Antibody 2A0G6 binds to Zika E in the fusion initiation state and blocks the exposure of the fusionpeptide

The structure of Zika E, bound with a broadly-protective antibody 2A0G6 (Fig. 3b) has been solved usingx-ray crystallography (PDB accession code: 5JHL) (51). The structure shows that the antibody interactswith Zika E residues 76, 77 and 105-108. These residues lie on the tip of the GP close to the fusionloop. In the pre-fusion state, these residues are buried and cannot be reached by the antibody, but in thefusion initiation state, these residues become exposed. When the antibody binds, the fusion loop (residues100-108) is blocked from exposure, and the fusion process is blocked.

Antibody EDE1-C8 blocks the pre-fusion to fusion transition of Zika E and prevents cell entry

The antibody (EDE1-C8) binds to the pre-fusion state structure of Zika E on the surface of the virus asshown in Fig. 4. With the antibody bound, it prevents the peeling off of the N-terminal regions (I, II andIII-1) of the Zika E protein from its neighboring Zika E and forming the fusion-state trimer structure. Withthe Zika E locked in the pre-fusion structure, it prevents the pre-fusion to fusion transition.

Videos describing the transitions from the pre-fusion to fusion structures

Large conformational changes are associated with the pre-fusion to fusion initiation state structures of theviral envelope protein. Even though the structures at both ends of the transition are known, it is not obvioushow to transform the structures from one to the other. To illustrate the complex process, two videos asan artists representation of the transition were developed. Each of the two videos depicts a possible (but

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antibody binding

site

2A0G6antibody binding

site

EDE1 C8

(a) (b)

Figure 4: Two different antibodies, 2A0G6 and EDE1 C8, interact with the Zika E to prevent viral cell entrywith different mechanisms. In this figure, the antibody recognition site is colored in red. a. Zika E in thepre-fusion state, the antibody-binding site is partially buried and prevents binding of 2A0G6 to E. b. ZikaE in the fusion initiation state, antibody-binding site is exposed, allowing 2A0G6 to bind to the E protein.The antibody EDE1 C8 binds to two copies of the Zika E and prevents transition from the pre-fusion tofusion initiation state.

not exclusive) transition pathway for Ebola GP and Zika E respectively. In these videos, the integrity ofstereochemistry was maintained for structures in each frame. Many of the features associated with thetransition and described in the manuscript are easily identifiable in the videos.

ConclusionsViral cell entry is a complex process involving multiple molecular participants and conformational changes.We provide a plausible mechanism in atomic detail for the pre-fusion/fusion-state transition for both Ebolaand Zika viruses. While these two viruses are very different, their fusion proteins share common features inthe initiation of cell entry. The most obvious common feature is a trimer arrangement of the proteins in thefusion initiation state. This can be seen in other viruses, such as Influenza and HIV. Secondly, these fusionproteins undergo large conformational changes to transition from pre-fusion to the fusion initiation statestructure. To prevent viral cell entry, antibody can act on the fusion protein using two different mechanisms:1) preventing the structural transition, and 2) blocking fusion peptide access to the cell membrane.

With the known structures of these proteins in the pre-fusion state and partial structures of the proteinsin the fusion initiation state, we developed near-complete fusion-state structural models of Ebola GP andZika E. Using a spring-loaded mechanism proposed for influenza hemagglutinin (HA) structural transition,the fusion initiation state trimer structure of the Ebola GP was developed. The fusion initiation state trimerstructure of the Zika E was developed from three dimers on the surface of the virus utilizing the three-foldsymmetry.

Combining the information of Ebola GP/Zika E structures in both the pre-fusion and the fusion ini-tiation states as well as the structures of these proteins in complex with different neutralizing antibodies,we investigated the mechanisms used by the antibody to prevent the process of viral cell entry. We foundthat KZ52 (Ebola) and EDE1-8 (Zika) prevented cell entry by blocking the pre-fusion to fusion structuraltransition. We proposed that the connecting bridge in the GP1 trimer (S90 to P93, P126 to R130) serves asa gate for the pre-fusion to fusion structural transition: if this gate is locked, the structural transition step isinhibited and the viral life cycle is halted. We also demonstrated that the antibody 2A0G6 binds to the ZikaE in the fusion initiation state and prevents the fusion peptide from reaching the cell membrane. The anti-body mAb100 is able to bind Ebola GP in both the pre-fusion- and the fusion initiation states, preventingthe viral cell entry both by inhibiting structural transition and by blocking fusion peptide access to the cellmembrane.

With the collective efforts to expand the information base in both biological sequences and structures,GenBank and PDB provide invaluable information. This information enables the expansion of structuralmodeling to a new realm that includes proteins in higher-order structure and/or transition. Using Ebola

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GP and Zika E as examples, we have demonstrated that a conceptually straightforward, knowledge-basedapproach allows us to develop models of the fusion initiation state structures for both proteins in theirfunctional units. With this information, we were able to study the binding of neutralizing antibodies tothese proteins and proposed the exact mechanisms for how these antibodies block cell entry to stop viralreplication.

Author contributionsAll authors were directly involved in regular discussions of model development and provided directionthroughout the project as well as participated in manuscript writing and editing. AL-V provided the energy-optimized structures of all the models developed from this work. She is also responsible for combiningdifferent edited versions into the final manuscript. WN developed the two videos that demonstrated thetransition of the two proteins from pre-fusion to fusion state. C-ST did the original structural modelingwork and provided oversight of the whole project.

AcknowledgmentsWe gratefully acknowledge the support of the U.S. Department of Energy through the LANL/LDRD Pro-gram, project number 20170509DR for this work. C-S T. is a LANL retired scientist and worked on thisproject on a pro bono basis. M.Z. acknowledges the funding of NIAID Centers of Excellence for InfluenzaResearch and Surveillance (CEIRS), contract number HHSN272201400004C.

SUPPLEMENTARY MATERIAL

Modeling fusion state structure of the Ebola GP using the spring-loaded mechanismThe crystal structures of the Ebola GP in fusion and pre-fusion states are shown as the top and the bottomimages in Fig. S1 respectively. Neither of the structures is complete: the pre-fusion GP structure (3CSY)is significantly more complete and includes large portions of GP1 (residues 31 to 310) and GP2 (residues502-595). Only partial structure of GP2 (residues 557 to 630) is solved and available to the public (2EBO).The color coding of the helical regions corresponds to the spring-loaded mechanism with the followingcolor assignments: green, 560-564; yellow, 565-582; magenta, 583-595.

In the fusion state structure of the GP trimer, the C-terminal regions (residues 596 to 603, highlightedin red) are wrapped around the triple-helices formed from the N-terminal regions (residues 557-595) ofthe three GP2 molecules. In the pre-fusion structure of the GP trimer, only a small portion of GP2, thetriple-helix formed from the magenta-colored helices, is internal to the GP1 trimer (shown in a space-fillingmodel in Fig. S1a). The remaining parts of GP2 are wrapped around the GP1 trimer from the outside. Withthe central cavity in the GP1 trimer (3CSY) being limited and only being able to accommodate a tight triplehelix, the C-terminal regions of GP2 (red helix in Fig. S1a) has to be ‘peeled’ from the central triple-helixand move to the space below the triplex. With this part of the molecule including the membrane proximalexternal region (MPER) and the transmembrane helix (TM), its main function should be anchoring theprotein to the surface of the virus. With insufficient information to model the structure of this portion of themolecule, we decide to leave this segment out of our model.

The common structure between the pre-fusion (3CSY) and fusion (2EBO) structures of GP2 is the triplehelix formed by segments (residues 583-595) boxed and color highlighted in magenta as shown in Fig. S1b.By matching the magenta-colored helices, the structure of the three helical segments (magenta, yellow,green) in 3CYS can be replaced by a single long helix from 2EBO. The N-terminal residues (514 to 564color-coded in cyan and green) of GP2 from 3CSY are added to the long helix by matching the greensegments of the helices (see Fig. S1c) resulting in the model of GP2 in the fusion state (see Fig. S1d).

Remodeling the fusion loop structureWith the modeled structure as shown in Fig. S1d, in a trimer arrangement, the fusion loops are pointingsideways instead of upwards to the host membrane. We decide to remodel this portion (residues 514 to 559)of GP2 in the fusion state structure. With the segment adopting a β-hairpin structure (see Fig. S2a), the task

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(a) (b) (c) (d)

Figure S1: a. Incomplete crystal structures of the Ebola GP in its pre-fusion state (above) and the fusionstate (below). b. Modeled triple helix formed by segments (residues 583-595) boxed and highlighted inmagenta. c. N-terminal residues (514 to 564 color-coded in cyan and green) of GP2 from 3CSY added tothe long helix by matching the green segments of the helices. d. The resulting complete model of the GP2in the fusion state.

becomes arranging three β-hairpins into a trefoil fold. By analyzing the protein database, we have learnedthat the structure of a β-hairpin trefoil fold can be found in the crystal structure of the cytolethal distendingtoxin (CDT) (PDB accession code: 2F2F, see the top image of Fig. S2a). By matching the two β-strands inthe GP2 hairpin to those in the trefoil fold, one can model the hairpin in a trefoil fold arrangement as shownin Fig. S2b. The NMR structure of the fusion loop (residues 525 to 538) has been solved and availablein PDB (accession code: 2RLJ and shown as the red molecule in Fig. S2c). We substituted this knownstructure of the fusion loop into the corresponding region in the hairpin (colored in blue in Fig. S2c) andobtained the remodeled structure of the hairpin as shown in Fig. S2d. The final model of the GP2 trimer inthe fusion state is shown in Fig. S2e.

Determination of Zika E domain III-1 placement in the fusion-state structureIt is obvious that domains I/II (residues 1 to 304, colored in cyan in Fig. S3a and Fig. S3b) need to rotate90◦ to transit from the pre-fusion to the fusion state. It is also clear that domain III-2 (residues 404 to 501,colored in green in both Fig. S3a and Fig. S3b) should serve as the anchor for the Zika E in the fusionstate and should be connected to other E proteins on the surface of the virus. Placing the domain III-1(residues 305-403) relative to other domains in Zika E when the protein is in the fusion state is a problemthat needed to be addressed: in the first case, when domains I/II move into the fusion state structure, domainIII-1 can move together with domains I/II (as shown in Fig. S3a); in the second case, domain III-1 can staywith domain III-2 and not move together with domains I/II (as shown in Fig. S3b). In these figures, theconnecting residues between domain II/III-1 and III-1/III-2 are highlighted as orange spheres. One can seethat in case one ( Fig. S3a), the distance between the two orange spheres is ∼ 50 A, which is too long toform a connection. If domain III-1 did not move together with domains I/II as in the second case, the twoorange spheres that connect domains II/III-1 and III-1/III-2 is separated with appropriate distances to form

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+

(a) (b)

(c) (d) (e)

Figure S2: a. Residues 514-559 (below). Protein database analysis shows that the structure of a β-hairpintrefoil fold can be found in the crystal structure of the cytolethal distending toxin (CDT) (above) b. Re-modeled residues 514-559 of GP2 in the fusion state structure adopt a β-hairpin structure. These threeβ-hairpins form a trefoil fold. c. The NMR structure of the fusion loop (residues 525 to 538) shown in red.The known structure of the fusion loop was substituted into the corresponding region in the hairpin (shownin blue) d. The remodeled structure of the hairpin. e. The resulting complete model of the GP2 in the fusionstate.

the connection. Based on this exercise, we determined that domain III-1 should stay with domain III-2while domains I and II is rotated into a new position when Zika E protein undergoes the pre-fusion to thefusion state transition.

Binding of EDE1 C8 to Zika E proteins on the surface of the virusWhile the crystal and solution environments are quite different, the known structures of various proteins inthe two different environments have shown similar structural folds (52). Here, we assume that the bindingmode derived from crystal structures of the protein/antibody complexes provides a stable interaction for thecomplex under physiological conditions. The crystal structure of EDE1 C8/Zika E complex has been solved(PDB accession code: 5LBS) and is shown (ribbon model) in Fig. S4a. We can use this binding mode totell how the antibody is going to interact with the Zika E when it is in its functional state (i. e when the Eproteins cover the entire viral surface). In this figure, three Zika Es in the functional state (PDB accessioncode: 5IRE) are shown in a space-filling model (blue, cyan and gray respectively). The task is to bring theZika E/antibody complex to the surface of the virus. When we match the Zika E in the Zika E/antibodycomplex (shown in Fig. S4a) to one of the Zika E monomers (colored in blue) on the surface of the virus,we can bring the antibody to the surface of the virus as shown in Fig. S4b. As a result of this exercise, wesee that EDE1 C8 is interacting with two E proteins (colored in blue and cyan) as shown in Fig. S4c.

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(a) (b)

III-1

III-2

III-1

III-2

I, II I, II

Figure S3: a. Zika E Domains I and II (residues 1 to 304) shown in cyan, domain III-1 (residues 305-403)shown in blue, domain III-2 (residues 404-501) shown in green. b. Zika E Domains I and II (residues 1 to304) shown in cyan, domain III-1 (residues 305-403) shown in red, domain III-2 (residues 404-501) shownin green.

AnimationVideo 1. A transition path of Ebola GP between pre-fusion and fusion states (left: side, right: top). Theintermediate structures were made by morphing and minimization. Each domain is colored as follows–GP1: yellow, red and blue, GP2: orange, cyan and green).Video 2. A transition path of Zika E between pre-fusion and fusion states (left: side view, right: top view).To clarify the structure, Zika domains were colored as follows– I: yellow, II– red, III-1: blue, III-2: cyan,and the remaining two units of the trimer orange and green. the remaining structures are colored in gray.

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(a)interacting with 2 ENVs

(b)

(c)

EDE1 C8

ENV

Figure S4: a. The crystal structure of EDE1 C8/Zika E complex (PDB accession code: 5LBS) is shown asa ribbon model. b. After matching Zika E in the virus/antibody complex to one of the Zika monomers onthe surface of the virus, the antibody can be positioned as shown. c. EDE1 C8 interacting with two Zika Es(colored in blue and cyan).

References1. Eckert, D. M., and a. P. S. Kim, 2001. Mechanisms of viral membrane fusion and its inhibition. Annu

Rev Biochem 70:777–810.

2. Kielian, M., and S. Jungerwirth, 1990. Mechanisms of enveloped virus entry into cells. Mol Biol Med7:17–31.

3. Dimitrov, D. S., 2004. Virus entry: molecular mechanisms and biomedical applications. Nat Rev Micro2:109–122.

4. Mas, V., and J. A. Melero, 2013. Entry of enveloped viruses into host cells: membrane fusion. Springer,Netherlands.

5. Sullivan, N., Y. Sun, Q. Sattentau, M. Thali, D. Wu, G. Denisova, J. Gershoni, J. Robinson, J. Moore,and J. Sodroski, 1998. CD4-induced conformational changes in the human immunodeficiency virusType 1 gp120 glycoprotein: consequences for virus entry and neutralization. J Virol 72:4694–4703.

6. Riedel, C., D. Vasishtan, C. A. Siebert, C. Whittle, M. J. Lehmann, W. Mothes, and K. Grunewald,2017. Native structure of a retroviral envelope protein and its conformational change upon interactionwith the target cell. J Struct Biol 197:172–180.

7. Tran, E. E. H., M. J. Borgnia, O. Kuybeda, D. M. Schauder, A. Bartesaghi, G. A. Frank, G. Sapiro,J. L. S. Milne, and S. Subramaniam, 2012. Structural Mechanism of Trimeric HIV-1 Envelope Glyco-protein Activation. PLOS Pathogens 8:1–18.

8. Wilen, C. B., J. Tilton, and R. Doms, 2012. HIV: cell binding and entry. Cold Spring Harb PerspectMed. 2.

13


http://dx.doi.org/10.1101/285130

9. Berman, H., J. Westbrook, Z. Feng, G. Gilliland, T. Bhat, H. Weissig, and P. Bourne, 2000. The ProteinData Bank. Nucleic Acids Res 28:235–242.

10. Ebola data and statistics, 2015, Accessed 2017-07-17. World Health Organization Situation sum-mary Data published on 31 July 2015. Http://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary-20150731?lang=en.

11. Dang, J., S. K. Tiwari, G. Lichinchi, Y. Qin, V. S. Patil, A. M. Eroshkin, and T. M. Rana, 2016. Zikavirus depletes neural progenitors in human cerebral organoids through activation of the innate immunereceptor TLR3. Cell Stem Cell 19:258–265.

12. Rasmussen, S. A., D. J. Jamieson, M. A. Honein, and L. R. Petersen, 2016. Zika virus and birth defectsreviewing the evidence for causality. N Engl J Med 374:1981–1987.

13. Huang, W.-C., R. Abraham, B.-S. Shim, H. Choe, and D. T. Page, 2016. Zika virus infection during theperiod of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild typemice. Sci Rep 6:34793.

14. Govero, J., P. Esakky, S. M. Scheaffer, E. Fernandez, A. Drury, D. J. Platt, M. J. Gorman, J. M. Richner,E. A. Caine, V. Salazar, K. H. Moley, and M. S. Diamond, 2016. Zika virus infection damages the testesin mice. Nature 540:438–442.

15. Souza, B. S. F., G. L. A. Sampaio, C. S. Pereira, G. S. Campos, S. I. Sardi, L. A. R. Freitas, C. P.Figueira, B. D. Paredes, C. K. V. Nonaka, C. M. Azevedo, V. P. C. Rocha, A. C. Bandeira, R. Mendez-Otero, R. R. Dos Santos, and M. B. P. Soares, 2016. Zika virus infection induces mitosis abnormalitiesand apoptotic cell death of human neural progenitor cells. Sci Rep 6:39775.

16. Zika data and statistics, 2017, Accessed 2017-07-17. World Health Organization Situation summaryData published on 10 March 2017. Http://www.who.int/emergencies/zika-virus/situation-report/10-march-2017/en/.

17. Quick, J., N. J. Loman, S. Duraffour, J. T. Simpson, E. Severi, L. Cowley, J. A. Bore, R. Koundouno,G. Dudas, A. Mikhail, N. Oudraogo, B. Afrough, A. Bah, J. H. J. Baum, B. Becker-Ziaja, J. P.Boettcher, M. Cabeza-Cabrerizo, A. Camino-Sanchez, L. L. Carter, J. Doerrbecker, T. Enkirch, I. G.Dorival, N. Hetzelt, J. Hinzmann, T. Holm, L. E. Kafetzopoulou, M. Koropogui, A. Kosgey, E. Kuisma,C. H. Logue, A. Mazzarelli, S. Meisel, M. Mertens, J. Michel, D. Ngabo, K. Nitzsche, E. Pallasch,L. V. Patrono, J. Portmann, J. G. Repits, N. Y. Rickett, A. Sachse, K. Singethan, I. Vitoriano, R. L.Yemanaberhan, E. G. Zekeng, T. Racine, A. Bello, A. A. Sall, O. Faye, O. Faye, N. Magassouba,C. V. Williams, V. Amburgey, L. Winona, E. Davis, J. Gerlach, F. Washington, V. Monteil, M. Jour-dain, M. Bererd, A. Camara, H. Somlare, A. Camara, M. Gerard, G. Bado, B. Baillet, D. Delaune, K. Y.Nebie, A. Diarra, Y. Savane, R. B. Pallawo, G. J. Gutierrez, N. Milhano, I. Roger, C. J. Williams, F. Yat-tara, K. Lewandowski, J. Taylor, P. Rachwal, D. J. Turner, G. Pollakis, J. A. Hiscox, D. A. Matthews,M. K. O. Shea, A. M. Johnston, D. Wilson, E. Hutley, E. Smit, A. Di Caro, R. Wlfel, K. Stoecker,E. Fleischmann, M. Gabriel, S. A. Weller, L. Koivogui, B. Diallo, S. Keita, A. Rambaut, P. Formenty,S. Gnther, and M. W. Carroll, 2016. Real-time, portable genome sequencing for Ebola surveillance.Nature 530:228–232.

18. Nanbo, A., M. Imai, S. Watanabe, T. Noda, K. Takahashi, G. Neumann, P. Halfmann, and Y. Kawaoka,2010. Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependentmanner. PLOS Pathogens 6:1–20.

19. Aleksandrowicz, P., A. Marzi, N. Biedenkopf, N. Beimforde, S. Becker, T. Hoenen, H. Feldmann,and H.-J. Schnittler, 2011. Ebola virus enters host cells by macropinocytosis and clathrin-mediatedendocytosis. J Infect Dis 204:S957.

20. Volchkov, V. E., H. Feldmann, V. A. Volchkova, and H.-D. Klenk, 1998. Processing of the Ebola virusglycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U S A 95:5762–5767.

21. Wang, J., B. Manicassamy, M. Caffrey, and L. Rong, 2011. Characterization of the receptor-bindingdomain of Ebola glycoprotein in viral entry. Virol Sin 26:156.

22. Manicassamy, B., J. Wang, H. Jiang, and L. Rong, 2005. Comprehensive analysis of Ebola Virus GP1in viral entry. J Virol 79:4793–4805.

14


http://dx.doi.org/10.1101/285130

23. Weissenhorn, W., A. Carfı, K.-H. Lee, J. J. Skehel, and D. C. Wiley, 1998. Crystal structure of theEbola Virus Membrane Fusion Subunit, GP2, from the Envelope Glycoprotein ectodomain. Mol Cell2:605–616.

24. Lee, J. E., M. L. Fusco, A. J. Hessell, W. B. Oswald, D. R. Burton, and E. O. Saphire, 2008. Structureof the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454:177–182.

25. Brister, J. R., Y. Bao, S. A. Zhdanov, Y. Ostapchuck, V. Chetvernin, B. Kiryutin, L. Zaslavsky,M. Kimelman, and T. A. Tatusova, 2014. Virus variation resourcerecent updates and future directions.Nucleic Acids Res 42:D660–D665.

26. Malashkevich, V. N., B. J. Schneider, M. L. McNally, M. A. Milhollen, J. X. Pang, and P. S. Kim, 1999.Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-A resolution. Proc Natl AcadSci U S A 96:2662–2667.

27. Dias, J. a. M., A. I. Kuehne, D. M. Abelson, S. Bale, A. C. Wong, P. Halfmann, M. A. Muhammad,M. L. Fusco, S. E. Zak, E. Kang, Y. Kawaoka, K. Chandran, J. M. Dye, and E. O. Saphire, 2011. Ashared structural solution for neutralizing Ebolaviruses. Nat Struct Mol Biol 18:1424–1427.

28. Bale, S., J. M. Dias, M. L. Fusco, T. Hashiguchi, A. C. Wong, T. Liu, A. I. Keuhne, S. Li, V. L. Woods,K. Chandran, J. M. Dye, and E. O. Saphire, 2012. Structural basis for differential neutralization ofEbolaviruses. Viruses 4:447–470.

29. Freitas, M. S., L. P. Gaspar, M. Lorenzoni, F. C. L. Almeida, L. W. Tinoco, M. S. Almeida, L. F.Maia, L. Degrve, A. P. Valente, and J. L. Silva, 2007. Structure of the Ebola Fusion Peptide in amembrane-mimetic environment and the interaction with lipid rafts. J Biol Chem 282:27306–27314.

30. Gregory, S. M., E. Harada, B. Liang, S. E. Delos, J. M. White, and L. K. Tamm, 2011. Structure andfunction of the complete internal fusion loop from Ebolavirus glycoprotein 2. Proc Natl Acad Sci U SA 108:11211–11216.

31. Murin, C. D., M. L. Fusco, Z. A. Bornholdt, X. Qiu, G. G. Olinger, L. Zeitlin, G. P. Kobinger, A. B.Ward, and E. O. Saphire, 2014. Structures of protective antibodies reveal sites of vulnerability on Ebolavirus. Proc Natl Acad Sci U S A 111:17182–17187.

32. Tran, E. E. H., J. A. Simmons, A. Bartesaghi, C. J. Shoemaker, E. Nelson, J. M. White, and S. Subra-maniam, 2014. Spatial localization of the Ebola Virus Glycoprotein Mucin-Like Domain determinedby Cryo-Electron Tomography. J Virol 88:10958–10962.

33. Malone, R. W., J. Homan, M. V. Callahan, J. Glasspool-Malone, L. Damodaran, A. D. B. Schneider,R. Zimler, J. Talton, R. R. Cobb, I. Ruzic, J. Smith-Gagen, D. Janies, J. Wilson, and Z. R. W. Group,2016. Zika virus: medical countermeasure development challenges. PLOS Neglected Tropical Diseases10:e0004530.

34. Cunha, M. S., D. L. A. Esposito, I. M. Rocco, A. Y. Maeda, F. G. S. Vasami, J. S. Nogueira, R. P.de Souza, A. Suzuki, M. Addas-Carvalho, M. d. L. Barjas-Castro, M. R. Resende, R. S. B. Stucchi,I. d. F. S. F. Boin, G. Katz, R. N. Angerami, and B. A. L. da Fonseca, 2016. First complete genome se-quence of Zika Virus (Flaviviridae, Flavivirus) from an autochthonous transmission in Brazil. GenomeAnnounc 4.

35. Kuhn, R. J., W. Zhang, M. G. Rossmann, S. V. Pletnev, J. Corver, E. Lenches, C. T. Jones, S. Mukhopad-hyay, P. R. Chipman, E. G. Strauss, T. S. Baker, and J. H. Strauss, 2002. Structure of Dengue Virus:implications for Flavivirus organization, maturation, and fusion. Cell 108:717–725.

36. Lindenbach, B. D., and C. M. Rice, 2003. Molecular biology of flaviviruses. Adv Virus Res 59:23–61.

37. Tung, C.-S., and B. H. McMahon, 2012. A structural model of the E. coli PhoB Dimer in the transcrip-tion initiation complex. BMC Struct Biol 12.

38. Meng, E., E. Pattersen, G. Souch, C. Huang, and T. Ferrin, 2006. Tools for integrated sequence-structure analysis with UCSF Chimera. BMC Bioinformatics 7.

39. Hess, B., C. Kutzner, D. van der Spoel, and E. Lindahl, 2008. GROMACS4: algorithms for highlyefficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4:435–447.

15


http://dx.doi.org/10.1101/285130

40. Pattersen, E., T. Goddard, C. Huang, G. Couch, D. Greenblatt, E. Meng, and T. Ferrin, 2004. UCSFChimera - a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612.

41. Carr, C. M., and P. S. Kim, 1993. A spring-loaded mechanism for the conformational change ofinfluenza hemagglutinin. Cell 73:823 – 832.

42. Carr, C. M., C. Chaudhry, and P. S. Kim, 1997. Influenza hemagluttinin is spring-loaded by a metastablenative conformation. Proc Natl Acad Sci U S A 94:14306–14313.

43. White, J. M., S. E. Delos, M. Brecher, and K. Schornberg, 2008. Structures and mechanisms of viralmembrane fusion proteins. Crit Rev Biochem Mol Biol 43:189–219.

44. Modis, Y., S. Ogata, D. Clements, and S. C. Harrison, 2004. Structure of the dengue virus envelopeprotein after membrane fusion. Nature 427:313–319.

45. Wang, H., Y. Shi, J. Song, J. Qi, G. Lu, J. Yan, and G. Gao, 2016. Ebola viral Glycoprotein bound toits endosomal receptor Niemann-Pick {C1}. Cell 164:258 – 268.

46. Dube, D., K. L. Schornberg, C. J. Shoemaker, S. E. Delos, T. S. Stantchev, K. A. Clouse, C. C. Broder,and J. M. White, 2010. Cell adhesion-dependent membrane trafficking of a binding partner for theebolavirus glycoprotein is a determinant of viral entry. Proc Natl Acad Sci U S A 107:16637–16642.

47. Stiasny, K., and F. X. Heinz, 2006. Flavivirus membrane fusion. J Gen Virol 87:2755–2766.

48. Stiasny, K., R. Fritz, K. Pangerl, and F. X. Heinz, 2011. Molecular mechanisms of flavivirus membranefusion. Amino Acids 41:1159–1163.

49. Moller-Tank, S., and W. Maury, 2015. Ebola Virus entry: a curious and complex series of events. PLOSPathogens 11:e1004731.

50. Misasi, J., M. S. A. Gilman, M. Kanekiyo, M. Gui, A. Cagigi, S. Mulangu, D. Corti, J. E. Ledgerwood,A. Lanzavecchia, J. Cunningham, J. J. Muyembe-Tamfun, U. Baxa, B. S. Graham, Y. Xiang, N. J.Sullivan, and J. S. McLellan, 2016. Structural and molecular basis for Ebola virus neutralization byprotective human antibodies. Science 351:1343–1346.

51. Dai, L., J. Song, X. Lu, Y.-Q. Deng, A. M. Musyoki, H. Cheng, Y. Zhang, Y. Yuan, H. Song, J. Hay-wood, H. Xiao, J. Yan, Y. Shi, C.-F. Qin, J. Qi, and G. Gao, 2016. Structures of the Zika Virus EnvelopeProtein and its complex with a flavivirus broadly protective antibody. Cell Host & Microbe 19:696 –704.

52. Sikic, K., S. Tomic, and O. Carugo, 2010. Systematic comparison of crystal and NMR protein structuresdeposited in the Protein Data Bank. Open Biochem J 4:83–95.

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List of Figures1 Structures of Ebola GP corresponding to the pre-fusion and fusion initiation states. a. (Top)

A trimer structure of GP1/GP2 in the pre-fusion state and (Bottom) GP2 in the fusion initia-tion state were solved using x-ray crystallography (PDB accession codes 3CSY and 2EBO,respectively). b. Model of the pre-fusion structure of the GP2 trimer with a spring-loadedmechanism. c. Hairpin loop structure (residues 510 to 559) of GP2, including the fusionpeptide (residues 524-539, red), remodeled into a conformation with the fusion peptidepointing toward the host cell surface. d. GP1/GP2 trimer in the fusion initiation state boundto the NPC1 receptor. Viral membrane is oriented at the bottom of each panel. . . . . . . . 4

2 The pre-fusion and fusion structures of the Zika E protein. a. The 3.8 Angstrom cryo-EMstructure of the Zika E dimer. b. Left Partial structure of DENV E, and Right modeledstructure of Zika E in their fusion initiation states. These structures are divided into fourdomains, highlighted as follows: (I: red, II: yellow, III-1: blue, III-2: cyan). c. The 180copies of Zika E cover (from cryo EM) the surface of the viral capsid, with intersectionpoints of 3- and 5-fold symmetries marked. d. A model of side- and top-views of the ZikaE trimer (marked as a spike) in the fusion initiation state. . . . . . . . . . . . . . . . . . . 6

3 The mAb100 antibody can bind to the EBOV GP trimer in both pre-fusion and fusioninitiation states. a. Modeled mAb100 binding to two GP2 monomers when the GP trimer isin the pre-fusion state, preventing the fusion initiation state transition. b. A post-transitionmodel, where the mAb100 binding site on GP2 is exposed at the top of the trimer, allowingthe antibody to bind. The model shows that up to three mAb100 antibodies can bind to theGP trimer in the fusion initiation state structure without steric interference. . . . . . . . . . 7

4 Two different antibodies, 2A0G6 and EDE1 C8, interact with the Zika E to prevent viralcell entry with different mechanisms. In this figure, the antibody recognition site is coloredin red. a. Zika E in the pre-fusion state, the antibody-binding site is partially buried andprevents binding of 2A0G6 to E. b. Zika E in the fusion initiation state, antibody-bindingsite is exposed, allowing 2A0G6 to bind to the E protein. The antibody EDE1 C8 binds totwo copies of the Zika E and prevents transition from the pre-fusion to fusion initiation state. 8

S1 a. Incomplete crystal structures of the Ebola GP in its pre-fusion state (above) and thefusion state (below). b. Modeled triple helix formed by segments (residues 583-595) boxedand highlighted in magenta. c. N-terminal residues (514 to 564 color-coded in cyan andgreen) of GP2 from 3CSY added to the long helix by matching the green segments of thehelices. d. The resulting complete model of the GP2 in the fusion state. . . . . . . . . . . 10

S2 a. Residues 514-559 (below). Protein database analysis shows that the structure of a β-hairpin trefoil fold can be found in the crystal structure of the cytolethal distending toxin(CDT) (above) b. Remodeled residues 514-559 of GP2 in the fusion state structure adopt aβ-hairpin structure. These three β-hairpins form a trefoil fold. c. The NMR structure of thefusion loop (residues 525 to 538) shown in red. The known structure of the fusion loop wassubstituted into the corresponding region in the hairpin (shown in blue) d. The remodeledstructure of the hairpin. e. The resulting complete model of the GP2 in the fusion state. . . 11

S3 a. Zika E Domains I and II (residues 1 to 304) shown in cyan, domain III-1 (residues 305-403) shown in blue, domain III-2 (residues 404-501) shown in green. b. Zika E DomainsI and II (residues 1 to 304) shown in cyan, domain III-1 (residues 305-403) shown in red,domain III-2 (residues 404-501) shown in green. . . . . . . . . . . . . . . . . . . . . . . 12

S4 a. The crystal structure of EDE1 C8/Zika E complex (PDB accession code: 5LBS) is shownas a ribbon model. b. After matching Zika E in the virus/antibody complex to one of theZika monomers on the surface of the virus, the antibody can be positioned as shown. c.EDE1 C8 interacting with two Zika Es (colored in blue and cyan). . . . . . . . . . . . . . 13

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abstract - biorxiv.org 20, 2018 · abstract membrane fusion ... j c miner , p w fenimore , w...

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