Project summary:

Structural description of protein-protein interactions is the cornerstone of knowledge enabling detailed molecular understanding of processes such as receptor-ligand recognition within the immune system, potentially leading to a design of targeted immunotherapeutics or drugs in general. Precise analysis of such interacting systems usually relies on X-ray structural analysis, which depends on the successful preparation of diffracting crystals of the given protein complex – a task significantly more difficult for flexible or large protein complexes/oligomers. On the contrary, biophysical methods such as AUC, ITC, SPR, MST, or small-angle X-ray scattering enable the study of protein interactions directly in solution, with cryoEM as a further option to assess the structure of large protein assemblies. A combination of these techniques will be used for detailed study and molecular description of interactions of selected human and rodent natural killer cell receptors with their cognate protein ligands (e.g., NKR-P1:LLT1, NKR-P1B:Clr-11, NKp30:B7-H6, NKp80:AICL). The affinity of some of these protein complexes is rather low, as is often the case in immune recognition, thus making them very challenging for structural studies. At the same time, we are interested in understanding how these interactions take place on the cell surface and trigger natural killer cell activation or how these receptor:ligand complexes assemble within the immune synapse on the membrane of a living cell. Therefore, super-resolution microscopy and single-molecule localization microscopy techniques, such as utilizing supported lipid bilayers to prepare reconstituted model membrane systems, will be employed, too.

Profile of an ideal candidate: MSc. or equivalent in Biochemistry, Molecular Biology, Immunology, or a related field, and a good knowledge of English is required. Experience with DNA cloning and recombinant protein expression and purification, protein-related biophysical and structural biology methods, flow cytometry and fluorescence microscopy, and cell-based immunological assays is of advantage.

Literature:

Bláha J., Skálová T., Kalousková B., Skořepa O., Cmunt D., Grobárová V., Pazicky S., Poláchová E., Abreu C., Stránský J., Kovaľ T., Dušková J., Zhao Y., Harlos K., Hašek J., Dohnálek J., Vaněk O. (2022): Structure of the human NK cell NKR-P1:LLT1 receptor:ligand complex reveals clustering in the immune synapse. Nat Commun 13, 5022. DOI: 10.1038/s41467-022-32577-6

Vaněk O., Kalousková B., Abreu C., Nejadebrahim S., Skořepa O. (2022): Natural killer cell-based strategies for immunotherapy of cancer. Adv Protein Chem Struct Biol 129, 91-133. DOI: 10.1016/bs.apcsb.2022.02.001

Kalousková B., Skořepa O., Cmunt D., Abreu C., Krejčová K., Bláha J., Sieglová I., Král V., Fábry M., Pola R., Pechar M., Vaněk O. (2021): Tumor marker B7-H6 bound to the coiled coil peptide-polymer conjugate enables targeted therapy by activating human natural killer cells. Biomedicines 9, 1597. DOI: 10.3390/biomedicines9111597

Skořepa O., Pažický S., Kalousková B., Bláha J., Abreu C., Ječmen T., Rosůlek M., Fish A., Sedivy A., Harlos K., Dohnálek J., Skálová T., Vaněk O. (2020): Natural killer cell activation receptor NKp30 oligomerization depends on its N-glycosylation. Cancers 12, 1998. DOI: 10.3390/cancers12071998

Vaněk O., Celadová P., Skořepa O., Bláha J., Kalousková B., Dvorská A., Poláchová E., Pucholtová H., Kavan D., Pompach P., Hofbauerová K., Kopecký V. Jr., Mesci A., Voigt S., Carlyle J. R. (2019): Production of recombinant soluble dimeric C-type lectin-like receptors of rat natural killer cells. Sci Rep 9, 17836. DOI: 10.1038/s41598-019-52114-8

Crystal structures of the extracellular domains of human LLT1 and CD69 receptors show the same conserved C-type lectin-like domain fold and non-covalent dimer formed through helix alpha 2.