An unexpected protein structure story

The structure of human NKR-P1 shows a unique dimerization interface. Panel (a) compares the crystal structures of the dimers of the NKR-P1 receptor-binding domain. Panel (b) shows a structural comparison between LLT1 (green) and NKR-P1 (cyan) dimers prepared by overlapping only one monomer from each dimer (middle). Although they share a similar structure, their contrast mode is just the opposite. Credit: Charles University

The discovery and quest for confirmation of a peculiar protein structure led to the description of the interaction of receptor groups on natural killer (NK) cells. The study by the research team of Dr. Ondřej Vaněk from the Department of Biochemistry at Charles University’s Faculty of Science and colleagues from the Institute of Biotechnology of the Czech Academy of Sciences at BIOCEV was recently published in the journal. Nature Communications.

The Immunological Recognition Structural Biochemistry Laboratory, led by Dr Ondig Fanuc, has produced an exciting story. It all began a few years ago with the observation of an unexpected protein structure for a future, and ended with a detailed description of specific structures and interactions of the immune system.

“We are interested in how the cells of the immune system recognize whether other cells in our body are healthy or unhealthy,” explains Dr. Vanek. His research team mainly focuses on natural killer cells, which are part of innate immunity, and if they sense that another cell in the body is not healthy, they can quickly eliminate it. Structural immunology here seeks to discover how receptors on the surface of immune cells recognize proteins (or other structures) on the surface of another cell. “These proteins tell the natural killer cell whether or not everything is OK. What happens in the end is not just an interaction between two proteins, it is the interaction of a number of interactions, and eventually an inhibitory or active signal will prevail,” Dr. Funk explains.

The study just published focuses on two types of proteins and their interaction. One of them is a receptor in natural killer cells called NKR-P1. This receptor is interesting because it acts as one of the main surface markers by which NK cells can be identified, although its structure is not yet known. NKR-P1 receptors are also found on the surface of some specific subpopulations of T lymphocytes, which are involved in many autoimmune diseases. However, in this context, its effect is not yet well characterized, and may change from a purely inhibitory to a calculated cost and thus contribute to the development of these diseases.

The second protein that the study focuses on is . recruit From the NKR-P1 receptor, a protein called LLT1. This protein is normally found in other cells of the immune system, and as Dr. Fanuc describes, “When the cells interact and touch each other’s surface, it makes them say they know each other and all is well.” However, the past 15 years of research have demonstrated that in many cases of cancer, LLT1 protein is expressed on the surface of cancer cells, where it inhibits immune response. Dr. adds. Vanek: “Unfortunately, the worse the tumor type, the higher the surface expression of LLT1 protein.” He and colleagues were the first to describe the structure of LLT1 in 2015.

This paper describes the two proteins and their interaction at several levels, from the atomic structure to the cellular level. The research team initially produced the proteins, crystallized them, and solved the structure of their complex.

“The result was totally unexpected and interesting. One wonders at that moment whether this is just a crystal artifact or whether such a structure is actually present on the cell surface,” notes Dr. Fanuc. The complex next step of the research was super-resolution microscopy, and the next stages of the study were performed on cell surface and live NK cells isolated from donor blood. By combining several methods, the research team verified previous observations in the crystal structure of the complex of both proteins and described the resulting functional consequences – under what conditions the NKR-P1 and LLT1 proteins must meet to produce an inhibitory signal.

Both the NKR-P1 receptor and its ligand LLT1 are homologous, that is, they always form pairs of two identical strands on the cell surface, connected by disulfide bonds. Until now, the idea was that when the two proteins interact, one dimer of the receptor binds one ligand dimer. However, thanks to the crystal structure of the NKR-P1 complex with LLT1, we know this is not true: the receptor half-dimer interacts with the half-dimer of the ligand, allowing the formation of binding groups for these molecules on the NK cell surface when it interacts with the target cell.

It took several years of research to test this hypothesis from the atomic to cellular level. The affinity of the studied proteins is very weak, and this is done only by gathering It became strong enough for the NK cell to sense the inhibitory signal. Thus, the necessity of multiple molecules converging is a kind of evolutionary protection against unnecessary or false stimuli, and thanks to the new study, we can see exactly how this interaction works at a structural level. This may aid the design of therapeutic proteins that can desirable influence the interaction between the immune system and cancer cells.

The study was conducted by Dr Ondig Vank’s team at Charles University’s Faculty of Science in collaboration with the team of Dr Jan Donalek from the Institute of Biotechnology of the Czech Academy of Sciences (BIOCEV), who was mainly involved in the structural analyses. Two researchers from Oxford University also contributed significantly to the research, performing crystallization measurements and X-ray diffraction.

“Several generations of students from our laboratory took part in this study, and the first author, Jean Blaha, obtained his Ph.D. in this research. Gradually, we learned more and more methods, and the students progressed a lot. Of them now they work in some of the best European research institutes”, Dr. Funk explains.

Jan Blaha, first author of the study and now a postdoctoral fellow at EMBL Hamburg, says, “The most interesting thing for me while working on this project was discovering new insights into relatively common data that led us to more complex experiments. I learned not to be afraid to follow my ideas. Crazy as long as it’s based on data. I’ve come to realize that many of the world’s experts are only human, and the most passionate are hilarious and willing to help with any crazy scientific idea.”

The immune system: first image of antigen-binding T-cell receptors at atomic resolution

more information:
Jean Blaha et al., Human NKR-P1 cytoskeleton: LLT1 receptor: a ligand complex that reveals clumps at the immune synapse, Nature Communications (2022). DOI: 10.1038 / s41467-022-32577-6

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