NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI- cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.

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

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

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 12840 Prague Czech Republic

Department of Biochemistry Oncode Institute Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands

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

Protein Technologies Vienna Biocenter Core Facilities GmbH Dr Bohr Gasse 3 1030 Vienna Austria

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Structure of the human NK cell NKR-P1:LLT1 receptor:ligand complex reveals clustering in the immune synapse

Tumor Marker B7-H6 Bound to the Coiled Coil Peptide-Polymer Conjugate Enables Targeted Therapy by Activating Human Natural Killer Cells