Virus Surface Glycoprotein Complexed with Fab KZ52 Antibody
Clay Brown '10 and Keith Miller '12
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
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
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.
are wrapped in a lipid bilayer that anchors the glycoprotein, or GP,
that project from the surface. In the center of the
virion is the tightly-wound RNA complexed with the proteins NP,
VP30, and L. Between the envelope and the nucleic acid lay
the viral proteins VP40 and VP24.
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
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
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.
GP actually exists as a trimer of three non-covalently attached
together form a
monomer is composed
of two linked subunits, with three GP2
formed by three GP1 subunits.
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).
trimerization is mediated by multiple GP2-GP2 and GP2-GP1 contacts,
with no major interactions between two GP1 subunits being recognized.
is responsible for
host cell attachment,
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.
is responsible for cell surface attachment
and can be divided into base,
base domain is composed of four discontinuous sections, which
form two mixed β-sheets with strands β3
between the two
domain also binds with
a helix of GP2
domain also contains
the aforementioned Cys 53.
head is located between the base and glycan
cap, and is also composed by four discontinuous segments that form a
mixed β-sheet (Note: β-8
is not encoded in pdb file)
by an α-helix
and a smaller,
stranded, antiparallel β sheet.
subdomain is stabilized by two disulfide bonds.
108-Cys 135 bond connects the surface
exposed loop (β8-β9)
the Cys 121-Cys 147
connection bridges the β8-β9
glycan cap complexes
with the head to form the inner edges of
the chalice bowl.
It is composed of an α-helix
against a four-stranded
also contains four predicted N-linked glycans at
Asn228, Asn238, Asn257 and Asn268.
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.
is responsible for the fusion of
host cell and viral membranes. It contains the internal
fusion loop, a distinct
hydrophobic patch that ultimately
with the target
membrane, as well as two heptad repeat regions
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
to 556 and uses an antiparallel
display a partly
helical hydrophobic fusion peptide
including residues Leu529, Trp531, Iso532, Pro533, Tyr534 and Phe535.
of these residues pack into the head region
of the neighboring GP1
antiparallel sheet is
covalently linked by a disulphide bond between Cys511 and
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
motif are disordered and cannot be resolved. However, post-fusion GP2
fragments demonstrate antiparallel
composed of the heptad repeats. The CX6CC
forms an intrasubunit disulphide bond between Cys601 and Cys608.
is a human antibody
isolated from a survivor from the 1995
outbreak of Ebola-Zaire virus in Kikwit, Democratic Republic of the
This antibody binds a
epitope at the base of the GP chalice, engaging three different
42-43 at the N terminus of GP1, and 505-514 and 549-556 at the N
antibody contacts a total of 15 residues by van der Waals interactions,
while forming direct hydrogen bonds with 8 residues. More
– H-bond with
Ser52 – H-bond with Asp552
Ser53 – H bond with Asp552
Pro97 – H-bond
Pro97 – Hydrophobic interactions with Cys556
Thr28 – Hydrophobic interactions with His549
– Hydrophobic interactions
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
– H-bond with Leu43 and hydrophobic interactions with Val42
– Hydrophobic interactions with Val42
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.
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.
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
Leu57, Leu63 and Arg64 are
important for fusion-mediated conformation changes.
to maintain the
Phe88, Phe153 and
His154 pack against the hydrophobic
residues from a neighboring internal fusion loop.
of the chalice,
Pro146 and Cys147,
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.
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,