Structural insights into two distinct nanobodies recognizing the same epitope of green fluorescent protein

Green fluorescent protein (GFP) and its derivatives are widely used in biomedical research, and the manipulation of GFP-tagged proteins by GFP-specific binders is highly desired. However, structural in-formation on how these binders bind with GFP is still lacking. In this study, we determined the crystal structure of the nanobody Nb2 complexed with superfolder GFP (sfGFP) at a resolution of 2.2 angstrom. Inter-estingly, although the complementarity-determining regions (CDRs) of Nb2 and LaG16 sequences were only 29.7% identical, they both bound to the same epitope of GFP and existed in the same orientation. Structural analysis indicated that they achieved similar binding characteristics through different mechanisms. We further verified the kinetics and thermodynamics of binding by biolayer interferometry (BLI) and isothermal titration calorimetry (ITC). Nb2 showed a slightly higher binding affinity for sfGFP than LaG16. The stability of GFP-specific nanobodies was verified by nano differential scanning fluo-rimetry (nanoDSF). Nb2 exhibited the highest melting temperature (Tm); thus, Nb2 is a promising GFP nanobody candidate for use in applications requiring harsh testing conditions. We also compared the binding sites of available GFP nanobodies and showed that some of them can simultaneously bind with GFP and assemble into multifunctional complexes to manipulate GFP-tagged target proteins. Our results provide atomic-scale binding information for Nb2-sfGFP, which is important for the further development of GFP-nanobody based fusion protein manipulation techniques. (c) 2021 Elsevier Inc. All rights reserved.

Automated summary

This study determined the crystal structure of the nanobody Nb2 complexed with superfolder GFP (sfGFP) and found that despite low sequence similarity with LaG16, they both bound to the same epitope of GFP. Nb2 showed slightly higher binding affinity for sfGFP than LaG16, and exhibited the highest melting temperature (Tm) among the tested GFP-specific nanobodies, making it a promising candidate for applications under harsh conditions. Additionally, some GFP nanobodies were shown to simultaneously bind with GFP and assemble into multifunctional complexes, providing atomic-scale binding information for further development of GFP-nanobody based fusion protein manipulation techniques.