Characterization and structure determination of a llama-derived nanobody targeting the J-base binding protein 1.

Abstract

J-base binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (β-D-glucosylhydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single-domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance screening identified Nb6 as a strong binder, recognizing JBP1 with a 1:1 stoichiometry and high affinity (K_d = 30 nM). Crystallization trials of JBP1 in complex with Nb6 yielded crystals that diffracted to 1.47 Å resolution. However, the dimensions of the asymmetric unit and molecular replacement with a nanobody structure clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in space group P3₁ with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64 Å. Ensemble refinement suggests that in the ligand-free state one of the complementarity-determining regions (CDRs) is flexible, while the other two adopt well defined conformations.