Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 12800 Prague Czech Republic

Department of Cell Biology Faculty of Science Charles University Viničná 7 12800 Prague Czech Republic

Department of Oncology Ludwig Institute for Cancer Research University of Lausanne Chemin des Boveresses 155 1066 Epalinges Switzerland

Division of Structural Biology Wellcome Centre for Human Genetics University of Oxford Roosevelt Drive OX3 7BN Oxford UK

EMBL Hamburg Unit c o DESY Notkestrasse 85 22607 Hamburg Germany

Institute of Applied Physics Biophysics group TU Wien Getreidemarkt 9 1060 Vienna Austria

Institute of Biotechnology The Czech Academy of Sciences BIOCEV Centre Průmyslová 595 25250 Vestec Czech Republic

School of Biological Sciences Nanyang Technological University Nanyang Drive 60 637551 Singapore Singapore

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