van Beusekom Bart, Heidebrecht Tatjana, Adamopoulos Athanassios, Fish Alexander, Pardon Els, Steyaert Jan, Joosten Robbie P, Perrakis Anastassis
Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
VIB-VUB Center for Structural Biology, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
Acta Crystallogr F Struct Biol Commun. 2018 Nov 1;74(Pt 11):690-695. doi: 10.1107/S2053230X18010282. Epub 2018 Oct 16.
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 = 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 P3 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.
J 碱基结合蛋白 1（JBP1）有助于碱基 J（β-D-葡糖基羟甲基尿嘧啶）的生物合成和维持，碱基 J 是一种仅限于某些原生动物的胸腺嘧啶修饰。制备了靶向 JBP1 的骆驼科（骆驼）单域抗体片段（纳米抗体），用作结晶伴侣分子。表面等离子体共振筛选确定 Nb6 为强结合剂，以 1:1 的化学计量比和高亲和力（K = 30 nM）识别 JBP1。JBP1 与 Nb6 复合物的结晶试验得到了衍射分辨率为 1.47 Å 的晶体。然而，不对称单元的尺寸以及用纳米抗体结构进行的分子置换清楚地表明，预期的与 JBP1 形成的复合物晶体实际上只是纳米抗体的晶体。Nb6 在空间群 P3 中结晶，不对称单元中有两个分子；其晶体结构精修至最终分辨率为 1.64 Å。整体精修表明，在无配体状态下，其中一个互补决定区（CDR）是灵活的，而另外两个则具有明确的构象。
