Structural Insights Into The Binding of Nanobodies LaM2 and LaM4 to the Red Fluorescent Protein mCherry

Red fluorescent proteins (RFPs) are widely used in molecular biology research, especially in deep tissues and animal models, because of their superior autofluorescence, light scattering, and phototoxicity to GFP. Although RFP can be easily monitored in vivo , improved manipulation of RFP is still desired. Using suitable nanobodies (Nbs) to bind to different epitopes of RFP is the most promising approach; thus, it is crucial to obtain structural information on how the different Nbs interact with RFP. We determined the crystal structures of the LaM2-mCherry and LaM4-mCherry complexes at 1.4 Å and 1.9 Å resolution. Our results showed that LaM2 binds to the side of the mCherry β-barrel, while Lam4 binds to the bottom of the β-barrel and does not interfere with the homo-oligomerization interface. The distinct binding sites of LaM2 and LaM4 were further verified by ITC, F-SEC and DLS assays. Our results also showed that LaM2 and LaM4 can bind simultaneously to mCherry, which is crucial for recruiting multiple operation elements to the RFP. The binding of LaM2 or LaM4 did not significantly change the chromophore environment of mCherry, which is important for fluorescence quantification assays, while several GFP Nbs significantly altered the fluorescence. Mutation of the residues of the LaM2 or LaM4 binding interface to mCherry significantly decreased the binding affinity of the Nb to mCherry. Our results provided atomic resolution interaction information on the binding of Nbs LaM2 and LaM4 binding with mCherry, which is important for developing detection and manipulation methods for RFP-based biotechnology.