Browsing by Subject "Cryo-EM"

COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has thus far claimed over six million lives. Vaccines against SARS-CoV-2 have successfully mitigated severe disease and eased the burden on healthcare systems. Neutralizing antibodies play crucial roles both in vaccine derived immunity, and as drugs widely utilized in monoclonal antibody therapy. Neutralizing antibodies primarily target the spike protein, where most of the neutralizing epitopes are located in the receptor binding domain (RBD). Elucidating the sites of vulnerability to neutralization on SARS-CoV-2 can facilitate the development of therapeutics. 7A12 is a newly-discovered IgG antigen-binding fragment (Fab) isolated from a COVID-19 patient in Finland that can neutralize SARS-CoV-2 by targeting the spike protein. In this thesis, a complex of the Fab 7A12 with SARS-CoV-2 spike ectodomain trimer was studied using cryogenic electron microscopy (cryo-EM) single-particle analysis to elucidate the epitope of 7A12 and to gain insight into the neutralization mechanism of 7A12. Cryo-EM data of the complex revealed that Fab 7A12 can bind to both “open” and “closed” conformations of RBD. Rigid-body fitting of the spike trimer and Fab 7A12 models into the cryo-EM density indicates that 7A12 recognizes an epitope in the RBD which is mainly located outside the ACE2 binding site. Together, these results suggest that the 7A12 epitope belongs to class III of SARS-CoV-2 neutralizing epitopes located in the RBD, indicating that 7A12 could neutralize by sterically hindering ACE2 and by preventing the adjacent RBD from changing to ”up” conformation. Furthermore, our results revealed an overlap of 7A12 epitope with the putative binding site of heparan sulfate, a host factor of SARS-CoV-2 infection, which might contribute to neutralization. 7A12-RBD interface mapping delineated the residues comprising the epitope, which do not include mutants found in Beta, Gamma, and Delta variants, while four mutants were found in Omicron within the epitope indicating that 7A12 is likely to neutralize Beta, Gamma, and Delta variants of concern.

Tiivistelmä – Referat – Abstract The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase is a master regulator of cell growth. In the presence of environmental cues, such as nutrients and growth factor, mTORC1 is transported to the lysosome where it is activated by a small GTPase Rheb. Dysregulation of mTORC1 has been linked to several diseases such as cancer and neurodegeneration. Despite our growing understanding of the nutrient-driven activation mechanism of mTORC1, we still do not fully understand how nutrients are transported out of the lysosome or how nutrient sensing is connected to nutrient transport. Recently, SLC38A9, a small lysosomal transmembrane protein, was identified as a mediator of the efflux of essential amino acids from the lysosome to the cytosol. It also acts as an amino acid sensor for mTORC1, playing a role in its activation. Due to poorly vascularized tumor cores, cancers such as pancreatic ductal adenocarcinoma, have access to very scarce amounts of free nutrients. Consequently, they rely on scavenging of protein macromolecules from the extracellular environment, followed by digestion inside lysosomes. The digested nutrients are released to the cytosol via transporters such as SLC38A9 and activate the mTORC1 pathway which carries out the growth processes. In fact, recent studies in mouse xenograft models have shown a severely slowed down growth of PDAC tumors with SLC38A9 knocked out. Blocking of SLC38A9 activity with pharmacologics or biologics would prevent the release of digested amino acids from the lysosomes, starving cancer cells of nutrients, while sparing normal cells that do not feed on extracellular proteins. However, SLC38A9 is still poorly understood, and development of selective inhibitors first requires mechanistic understanding of the protein and knowing what its binding pockets look like. In order to obtain this information, we aimed to determine the three-dimensional structure of SLC38A9 through cryogenic electron microscopy (cryo-EM). However, two significant challenges hindered our ability to obtain high-resolution images of this membrane protein: (i) its small size, and (ii) its constant conformational changes. To address this, I proceeded to develop a set of nanobodies that would bind SLC38A9 with high affinity and specificity. Nanobodies allow for locking of target proteins in specific conformational states, and they can also serve as chaperones for visualizing proteins in cryo-EM. To obtain these nanobodies, I used a library of 100 million unique nanobodies, displayed on the surface of yeast cells. Specific SLC38A9 binder nanobodies were obtained through multiple rounds of selection and sorting, using decreasing concentrations of fluorescently- labeled SLC38A9. After the final selection round, single colonies were picked and the strength of binding to SLC38A9 was evaluated. High-throughput screening results showed that we were able to obtain specific SLC38A9 binders and that there was variation in binding strength among the selected nanobodies. These nanobodies will enable the determination of the cryo-EM structure of SLC38A9 and also serve as tools to further dissect the function and mechanisms of SLC38A9 in amino-acid efflux from lysosomes to cytosol, providing further insights for the development of novel cancer therapeutics.