STRUCTURAL BIOLOGY

38 ESRF

(Figure 23c), confirming the domain to be a pentamer in solution. The pentamer was found to be highly resistant to salt, temperature and the presence of denaturing agents. The surface charge distribution of the pentamer revealed a higher net negative charge on its proximal face, suggesting a strong histone interaction potential. The pentamer was found to interact strongly with histone H2A/H2B dimer and H3/H4 tetramer in a 1:1 stoichiometry (Figure 23d), both individually as well as simultaneously, suggesting separate binding sites for the histone oligomers on the pentamer. The ability of the pentamer to perform nucleosome assembly was confirmed through a plasmid supercoiling assay (Figure 23e). Further investigation of its interaction with preformed nucleosomes showed two pentamers to form

a complex with the nucleosome. The crystal structure of the C-terminal domain (CTD) in complex with FK506 revealed a monomeric structure, characteristic of FKBPs, having a five-beta palm-like fold wrapping over helices (Figure 24a), and the CTD was confirmed to be an active peptidyl-prolyl isomerase (Figure 24b).

This forms the first structural and functional characterisation report of a plant nucleoplasmin. The protein takes up a pentapus-like structure with a pentameric core, leading to five long arms and terminal PPIase domains. This work is expected to form the base for additional studies towards the identification and structural elucidation of physiologically relevant complexes of plant nucleoplasmin-FKBPs.

Fig. 23: a) The crystal structure of AtFKBP53 NTD pentamer. b) Structural features of the AtFKBP53 NTD monomer. c) The AtFKBP53 NTD molecular envelope generated from the SAXS experimental data in P5 symmetry with the crystal structure

fitted inside. d) Isothermal titration calorimetric data for the interaction of AtFKBP53 NTD with histone H2A/H2B (blue) and H3/H4 (red). e) Plasmid supercoiling assay showing the efficiency of AtFKBP53 NTD to perform nucleosome assembly.

Fig. 24: a) The crystal structure of AtFKBP53 CTD in complex with FK506. b) Chymotrypsin-coupled PPIase assay for AtFKBP53 CTD showing an increase in the absorbance as a function of time, indicating PPIase activity and a faster rate with increasing amounts of the protein.