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

Abstract

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.

Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies

“…Nb GFP employs all three CDR regions for antigen binding (PDB ID: 3OGO) 20 . Instead, in the LaM4 interaction with mCherry, the binding occurs via the CDR3 and only partly via CDR1 (PDB ID: 6IR1) 48 . Most of the contacting surfaces of the Nb6 and spike SARS-CoV-2 RBD (receptor binding domain) are contributed by the CDR1 and CDR2 47 .…”

Development of AlissAID system targeting GFP or mCherry fusion protein

“…Considering that the GFP-banding force of VHHGFP4 has a KD value of 1.40 nM (37), degradation efficiency appears to be associated with the affinity to the nanobody and target proteins. In terms of crystal structures, the mCherry LaM2 binding site is similar to the GFP binding site, which VHHGFP4 binds to (36,37). These binding sites may potentially be related to efficient ubiquitination and degradation.…”

“…Among them, LaM2 induced target protein degradation most efficiently, whereas LaM4 weakly induced target degradation. In terms of mCherry-binding forces, LaM2 and LaM4 have KD values of 3.02 ± 1.88 nM and 22.50 ± 34.6 nM, respectively (36). Considering that the GFP-banding force of VHHGFP4 has a KD value of 1.40 nM (37), degradation efficiency appears to be associated with the affinity to the nanobody and target proteins.…”

Numaswitch, a biochemical platform for the efficient production of disulfide-rich pepteins

“…For both microorganisms MIC values of 1.6 µM were determined, being consistent with previous reported values (Chen et al, 2021;Dash and Bhattacharjya, 2021) and demonstrating the production of functional target (Supplementary Figure S7). After co-incubation of VHH, a single chain antibody fragment with binding affinity towards the fluorescent protein mCherry (Wang et al, 2021), the quantitative formation of VHH-mCherry heterodimers was observed by size exclusion chromatography (SEC) analysis (Supplementary Figure S8), proving the functional fold of VHH. Activity of the recombinantly produced anti-tubulin scFv was analyzed by epifluorescence imaging (Nizak et al, 2003).…”