Pursuit of structural mechanisms to explain extraordinary pathogenesis
Our laboratory seeks to understand at the molecular level how certain pathogens overcome and even exploit the human immune system. We begin by determining an X-ray crystal structure of a key pathogenic determinant or immune molecule, and then use the structure to develop hypotheses which we subsequently test through immunological, biochemical and functional studies. Our goal is to marry structural and functional studies on key pathogens within the same laboratory by the same researchers.

Ebolavirus: Two glycoprotein structures from the same gene: The ebolavirus is a negative-stranded, membrane-enveloped filovirus which causes a 50-90% lethal hemorrhagic fever. There are five species of the virus, of which Zaire and Sudan are the most common and most pathogenic. Interestingly, the ebolavirus makes two different forms of the viral glycoprotein from the same GPgene. These two proteins share an identical 295 amino acid N-terminal region, but encode different C termini as a result of transcriptional editing. The different C termini lead to unique patterns of disulfide bonding and different structures. Importantly, it is the different structures that lead to different roles in pathogenesis.

The rarer of these two proteins is a ~450 kDa trimeric, viral surface glycoprotein termed GP. GP is critical in the viral life cycle as it is solely responsible for attachment, fusion and entry of target cells. Interestingly, most of the antibodies raised in natural infection are directed towards sGP rather than GP. Hence, antibodies that neutralize the virus can be difficult to elicit in natural infection and many survivors have low to insignificant titers.Trimeric, prefusion, Zaire ebolavirus GP: We have recently published a crystal structure of the Zaire ebolavirus glycoprotein in its trimeric, prefusion form in complex with KZ52, a neutralizing antibody from a human survivor. Structures of viral glycoproteins in their native, viral surface forms can be difficult to achieve owing to the heavily glycosylated, oligomeric, metastable nature of these proteins. Indeed, we had to grow ~50,000 crystals for this project, and screen the 800 largest crystals over ~30 synchrotron trips in order to find one crystal that would diffract to 3.4 Å and permit structure determination. Importantly, the GP crystallized retains all regions required for attachment, fusion and entry. Viruses pseudotyped with our crystallization construct plus the transmembrane domain are functional in infectivity assays and exhibit antibody neutralization profiles identical to wild-type GP. Our crystal structure illustrates that trimeric GP forms a three-lobed chalice shape with the bowl of the chalice assembled by the three GP1 attachment subunits. The stem of the chalice is formed by three GP2 fusion subunits that cradle and encircle the GP1 trimer. Here, the internal fusion loop and heptad repeat region of GP2 together wrap around GP1, and in turn, hydrophobic residues of GP1 clamp the heptad repeat of GP2 into its metastable, prefusion conformation. This clamp is released in entry through an as yet unidentified process, allowing GP2 to spring into its six-helix bundle conformation and trigger fusion of virus and host membranes. CLICK HERE TO VIEW MOVIE

Additional structural targets in the lab aim to elucidate mechanisms of innate immune suppression, viral assembly and replication of the ebola virus, as well as glycoprotein architecture of other hemorrhagic fever viruses as well.