Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein–protein interactions have been performed, the very weak interactions generally observed in cell–cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (Kd greater than μM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 μM (HLA-Cw4) and 5.7 μM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell–cell recognition receptors involved in immunologically relevant events.