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

Abstract

The J-base Binding Protein 1 (JBP1) contributes to biosynthesis and maintenance of base J ({beta}-D-glucosyl-hydroxymethyluracil), 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 (SPR) screening identified Nb6 as a strong binder, recognising JBP1 with a 1:1 stoichiometry and high affinity (kD=30nM). Crystallisation trials of JBP1 in complex with Nb6, yielded crystals diffracting to 1.47[A] resolution.\n\nHowever, the asymmetric unit dimensions 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 spacegroup P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64[A]. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.\n\nSynopsisA camelid single domain antibody fragment (nanobody) is shown to have high affinity towards its recognition target, the J-base binding protein 1 (JBP1). The serendipitous crystallisation of this nanobody alone, and its crystal structure solution and refinement to 1.64[A] resolution are described. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.