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1.  Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus 
Journal of Virology  2012;86(5):2809-2816.
Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.
doi:10.1128/JVI.05549-11
PMCID: PMC3302272  PMID: 22171276
2.  Cathepsin Cleavage Potentiates the Ebola Virus Glycoprotein To Undergo a Subsequent Fusion-Relevant Conformational Change 
Journal of Virology  2012;86(1):364-372.
Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ∼10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.
doi:10.1128/JVI.05708-11
PMCID: PMC3255896  PMID: 22031933
3.  A Shared Structural Solution for Neutralizing Ebolaviruses 
Nature structural & molecular biology  2011;18(12):1424-1427.
Sudan virus (genus ebolavirus) is lethal, yet no monoclonal antibody is known to neutralize it. Here we describe antibody 16F6 that neutralizes Sudan virus and present its structure bound to the trimeric viral glycoprotein. Unexpectedly, the 16F6 epitope overlaps that of KZ52, the only other antibody against the GP1,2 core to be visualized. Further, both antibodies against this key GP1–GP2-bridging epitope neutralize at a post-internalization step, likely fusion.
doi:10.1038/nsmb.2150
PMCID: PMC3230659  PMID: 22101933
4.  Structural Basis for Differential Neutralization of Ebolaviruses 
Viruses  2012;4(4):447-470.
There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP1,2. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP1,2 molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP1,2 from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP1-GP2 clamp, thermal motion of various regions in GP1,2 across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization.
doi:10.3390/v4040447
PMCID: PMC3347318  PMID: 22590681
Filovirus; Ebola; ebolavirus; Sudan virus; neutralization: glycoprotein; antibodies; structure
5.  Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein 
Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described.
The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glyco­protein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.
doi:10.1107/S0907444909032314
PMCID: PMC2777170  PMID: 19923712
glycoproteins; structure determination; difficult structures; antibody complexes; viral proteins; human proteins; tactics to improve diffraction; techniques for phase determination; deglycosylation; model building
6.  An efficient platform for screening expression and crystallization of glycoproteins produced in human cells 
Nature protocols  2009;4(4):592-604.
Glycoproteins mediate multiple, diverse and critical cellular functions, that are desirable to explore by structural analysis. However, structure determination of these molecules has been hindered by difficulties expressing milligram quantities of stable, homogeneous protein and in determining, which modifications will yield samples amenable to structural studies. We describe a platform proven effective for rapidly screening expression and crystallization of challenging glycoprotein targets produced in mammalian cells. Here, multiple glycoprotein constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and subsequently screened in small quantities for crystallization by microfluidic free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be screened for expression and crystallization and scaled up for structural studies in a matter of five weeks.
doi:10.1038/nprot.2009.29
PMCID: PMC2911120  PMID: 19373230
7.  Structure of the Ebola virus glycoprotein bound to a human survivor antibody 
Nature  2008;454(7201):177-182.
Ebola virus (EBOV) entry requires the surface glycoprotein, GP, to initiate attachment and fusion of viral and host membranes. Here, we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the N terminus of GP1. This structure now provides a template for unraveling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.
doi:10.1038/nature07082
PMCID: PMC2700032  PMID: 18615077
8.  Complex of a protective antibody with its Ebola virus GP peptide epitope: unusual features of a Vλx-light chain 
Journal of molecular biology  2007;375(1):202-216.
13F6-1-2 is a murine monoclonal antibody that recognizes the heavily glycosylated mucin-like domain of the Ebola virus virion-attached glycoprotein (GP) and protects animals against lethal viral challenge. Here we present the crystal structure, at 2.0 Å, of 13F6-1-2 in complex with its Ebola virus GP peptide epitope. The GP peptide binds in an extended conformation, anchored primarily by interactions to the heavy chain. Two GP residues, Gln P406 and Arg P409, make extensive side chain hydrogen bond and electrostatic interactions to the antibody and are likely critical for recognition and affinity. The 13F6-1-2 antibody utilizes a rare Vλx light chain. The three light chain complementarity determining regions (CDRs) do not adopt canonical conformations and represent new classes of structures distinct from Vκ and other Vλ light chains. In addition, although Vλx had been thought to confer specificity, all light chain contacts are mediated through germline-encoded residues. This structure of an antibody that protects against the Ebola virus now provides a framework for humanization and development of a post-exposure immunotherapeutic.
doi:10.1016/j.jmb.2007.10.017
PMCID: PMC2173910  PMID: 18005986
Ebola virus; Vλx light chain; glycoprotein; neutralizing antibody; new canonical structures of immunoglobulins; hypervariable loops; complementarity determining region; Fab-peptide complex

Results 1-8 (8)