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

Acta Crystallogr F Struct Biol Commun. 2018 Nov 1;74(Pt 11):690-695. doi: 10.1107/S2053230X18010282. Epub 2018 Oct 16.

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 (Kd = 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.

Keywords: J-base binding protein 1; immune system; llamas; nanobodies.

MeSH terms

  • Animals
  • Camelids, New World
  • Crystallography, X-Ray
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Glucosides / metabolism
  • Models, Molecular
  • Protein Conformation
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / metabolism*
  • Single-Domain Antibodies / chemistry*
  • Single-Domain Antibodies / genetics
  • Single-Domain Antibodies / immunology
  • Single-Domain Antibodies / metabolism*
  • Surface Plasmon Resonance
  • Uracil / analogs & derivatives
  • Uracil / metabolism

Substances

  • DNA-Binding Proteins
  • Glucosides
  • J-specific DNA-binding protein, protozoa
  • Protozoan Proteins
  • Single-Domain Antibodies
  • 5-((glucopyranosyloxy)methyl)uracil
  • Uracil