Understanding the nanoscale organization of natural killer cell receptor NKp30 at the single- molecule level

Abstract

The NKp30 receptor is a molecule that activates natural killer (NK) cell cytotoxicity towards malignant or infected cells by recognizing markers of poor cell health, such as the stress-induced molecule B7-H6. This receptor's ability to identify and eliminate unhealthy cells makes it a target for various immunotherapeutic strategies. The efficiency of NK cell activation depends on ligand recognition and successful signal transduction, necessitating a deep understanding of the structural features involved in ligand recognition for innovative and effective immunotherapeutic design. Previous studies have shown that the NKp30 ligand binding correlates with its oligomeric state (Hartmann et al., 2012; Herrmann et al., 2014). In turn, the oligomeric formation depends on its N-glycosylation and the presence of the proximal membrane region (stalk domain). Moreover, the crystal structure of the NKp30:B7-H6 complex (PDB ID 6YJP) emphasizes the importance of Asn42 glycosylation (Skořepa et al., 2020). For the research presented, we employ single-molecule fluorescence microscopy methods to link NKp30 structural data with the observations on the cell surface or in 2D model systems using functionalized supported lipid bilayers as membrane surrogates. This approach aims to uncover the biological relevance of NKp30 oligomerization. We utilize single-molecule tracking and the TOCCSL approach (thinning out clusters while conserving the stoichiometry of labeling, Moertelmaier et al., 2005) to comparatively analyze different NKp30 sequence variants, including glycomutants and stalk-deficient constructs. NKp30-specific nanobodies or affinity- matured ligand B7H6 serve asfluorescentprobesinourstudies.