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Ebola Virus Surface Glycoprotein Complexed with Fab KZ52 Antibody

Clay Brown '10 and Keith Miller '12


I. Introduction

The Ebola virus (EBOV) is an enveloped, negative-sense RNA virus that, along with the Marburg virus, makes up the Filoviridae family. Although it first emerged in 1976 in Zaire, it has been making waves in the epidemiological world because of its 50-90% human mortality rate. Initial symptoms such as fever, exhaustion, headache and nausea gradually give way to massive internal and external hemorrhage in nearly every organ of the body as the virus interferes with the endothelial cells lining the internal surfaces of blood vessels. If patients do not recover by the second week of infection, death from either hypovolemic shock or multisystem organ failure is to be expected. Luckily, the virus is mainly spread by bodily fluids, which has helped to prevent an epidemic.

Electron micrographs have shown that the Ebola virus has the characteristic thread or shepherd's crook shape of a filovirus. Virions are wrapped in a lipid bilayer that anchors the glycoprotein, or GP, spikes that project from the surface. In the center of the virion is the tightly-wound RNA complexed with the proteins NP, VP35, VP30, and L. Between the envelope and the nucleic acid lay the viral proteins VP40 and VP24.

The Ebola genome encodes seven genes that code for eight proteins. Editing of the GP gene can result in either a truncated 364-residue secreted glycoprotein (sGP) or a full length 676-residue structural glycoprotein (GP) that is further cleaved to yield disulphide linked GP1 and GP2 subunits. It is EBOV GP that helps mediate viral entry into the cell while also retarding immune response. Thus, it is unique that this particular protein crystal includes the Fab KZ52 antibody from a human survivor.   This structure helps to explain why antibodies that neutralize the virus are so rare, yet also identifies the few sites that an antibody or vaccine could possibly bind.

II. Pre-Fusion Conformation

EBOV GP actually exists as a trimer of three non-covalently attached monomers (A, B and C ) that together form a structure not unlike a chalice. Each monomer is composed of two linked subunits, with three GP2 domains  encircling the bowl formed by three GP1 subunits. These units are connected by a disulfide bridge between Cys 53 of EBOV GP1 and Cys 609 of the GP2 subunit (Note: Cys 609 is not encoded in pdb file).  GP trimerization is mediated by multiple GP2-GP2 and GP2-GP1 contacts, with no major interactions between two GP1 subunits being recognized.  

GP1 is responsible for host cell attachment, while GP2 allows for fusion of the viral and cell membranes. The Ebola virus enters host cells via receptor-mediated endocytosis at specific clathrin coated pits or lipid rafts through a process involving an irreversible conformation change in GP that causes merging of viral and cell membranes. The structure of EBOV GP examined here corresponds to the pre-fusion conformation of the protein as determined by the orientation of the GP2 subunit.


GP1 is responsible for cell surface attachment and can be divided into base, head and glycan cap regions.   The base domain is composed of four discontinuous sections, which form two mixed β-sheets with strands β3 and β13   shared between the two sheets.   This domain also binds with a helix of GP2 through hydrophobic interactions.  This domain also contains the aforementioned Cys 53.

The head is located between the base and glycan cap, and is also composed by four discontinuous segments that form a four-stranded, mixed β-sheet (Note: β-8 is not encoded in pdb file)
supported by an α-helix and a smaller, two stranded, antiparallel β sheet.  This subdomain is stabilized by two disulfide bonds.  The Cys 108-Cys 135 bond connects the surface exposed loop (β8-β9) to strand β7,   while the Cys 121-Cys 147 connection bridges the β8-β9 and β9-β10 loops.

The glycan cap complexes with the head to form the inner edges of the chalice bowl.  It is composed of an α-helix packed against a four-stranded β-sheet.  This region also contains four predicted N-linked glycans at Asn228, Asn238, Asn257 and Asn268.  These oligosaccharides help to conceal the sides and top of the chalice, which proves key in the prevention of antibody and inhibitor binding. The glycan cap also contacts a serine-threonine-rich, mucin-like domain that has been proven to be responsible for the cytotoxic effects in human endothelial cells. Unfortunately, this domain was excised in order to increase to sample homogeneity and to promote crystal contacts in this crystallization experiment.


GP2 is responsible for the fusion of host cell and viral membranes.  It contains the internal fusion loop, a distinct hydrophobic patch that ultimately interacts with the target membrane, as well as two heptad repeat regions designated HR1 and HR2.

While most viruses have fusion peptides located at the N terminus of their fusion subunit, the Ebola virus GP contains an internal fusion loop lacking this free terminus. This loop encompasses residues 511 to 556 and uses an antiparallel 
β-stranded scaffold to display a partly helical hydrophobic fusion peptide including residues Leu529, Trp531, Iso532, Pro533, Tyr534 and Phe535. The side chains of these residues pack into the head region of the neighboring GP1 subunit.  The antiparallel sheet is covalently linked by a disulphide bond between Cys511 and Cys556.    

EBOV GP2 also contains two heptad repeat regions that are connected by a 25-residue linker containing a CX6CC motif and the internal fusion loop. In the pre-fusion conformation of GP, HR2 and the CX6CC motif are disordered and cannot be resolved. However, post-fusion GP2 fragments demonstrate antiparallel α-helices composed of the heptad repeats. The CX6CC motif forms an intrasubunit disulphide bond between Cys601 and Cys608.  

V. The Human Antibody KZ52 

KZ52 is a human antibody isolated from a survivor from the 1995 outbreak of Ebola-Zaire virus in Kikwit, Democratic Republic of the Congo.  This antibody binds a non-glycosylated epitope at the base of the GP chalice, engaging three different segments: residues 42-43 at the N terminus of GP1, and 505-514 and 549-556 at the N terminus of GP2.   The antibody contacts a total of 15 residues by van der Waals interactions, while forming direct hydrogen bonds with 8 residues. More specifically, the interactions include:

Thr100 – H-bond with Gly553
Ser52 – H-bond with Asp552
Ser53 – H bond with Asp552
Pro97 – H-bond with Asn550
Pro97 – Hydrophobic interactions with Cys556
Thr28 – Hydrophobic interactions with His549

Tyr32 – Hydrophobic interactions with Pro513
Trp50 – Hydrophobic interactions with Gln508 and Ala 507
Lys30 – Hydrophobic interactions with Ala507
Asn28 – Hydrophobic interactions with Ala507 and Val505
Arg98 – H-bonds with Pro509 and Gln508
Asn31 – H-bond with Asn514

Tyr100 – H-bond with Leu43 and hydrophobic interactions with Val42
Tyr56 – Hydrophobic interactions with Val42

Of the 15 GP residues that contact the KZ52 antibody, 10 are unique to the Zaire ebolavirus strain, thus explaining the specificity of KZ52. KZ52 likely neutralizes cytoxicity of Ebola by blocking the membrane insertion of the internal fusion loop. It may also block access to the receptor binding site. Interestingly, the presence of both GP1 and GP2 is necessary for KZ52 recognition. It is possible that GP1 is needed to hold GP2 in the proper pre-fusion structure for KZ52 to bind.  

However, the development of neutralizing antibodies in those infected with Ebola is limited. The glycan cap is topped with both a mucin-like domain and several oligosaccharides. This glycocalyx surrounds EBOV GP and forms a shield that protects the protein from humoral immune response. 

VI. Receptor Binding 

Although essential cellular receptors for the Ebola virus have not been identified, previous studies have indicated that residues 54-201 of the base and head subunits form a domain that attaches to host cells. In particular, 19 GP1 residues, most of which are involved in maintaining the structural integrity of GP1, have been identified as critical for viral entry. 

Asp55, Leu57, Leu63 and Arg64 are important for fusion-mediated conformation changes.

Lys95, Phe159, Phe160, Tyr162 and Ile170 help to maintain the structural stability of GP1.

Gly87, Phe88, Phe153 and His154 pack against the hydrophobic residues from a neighboring internal fusion loop.

Six residues in the inner bowl of the chalice, Lys114, Lys115, Lys140, Gly143, Pro146 and Cys147, represent important receptor binding contact sites.  

Oddly, these receptor binding contact sites are masked by both the glycan cap and the mucin-like domain. This suggests that additional conformation changes or the removal of certain domains may be required for receptor binding. It has been demonstrated that cathepsin L and/or B are able to remove the mucin-like domain, while possibly removing the entire glycan cap as well. If this occurred, then the important binding sites would become exposed and could interact with the elusive Ebola receptor.

VII. References

Lee, J. E., Fusco, M. L., Hessell, A. J., Oswald, W. B., Burton, D. R. & Saphire, E. O. (2008). Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature, 454, 177-181.

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