Comparison of PfNapL structure with yNAP-1 (Protein Data Bank code 2AYU) and hSET (Protein Data Bank code 2E50). a, superimposition of PfNapL monomer (purple) onto yNAP-1 (blue) and hSET (orange). Accessory domain of yNAP-1 is colored yellow. NES and NLS in yNAP-1 and PfNapL are indicated. b, superimposition of PfNapL and yNAP-1 dimers. The lower half shows superimposition of domain I and II from PfNapL and yNAP-1 monomers. yNAP-1 is colored blue, and the accessory domain is colored yellow. PfNapL is colored purple. The extra residues in domain II of yNAP-1 are colored cyan and are disordered in PfNapL. c, superimposition of PfNapL and hSET dimers. The lower half shows superimposition of domain I and II from PfNapL and hSET monomers. The extra residues of PfNapL are colored cyan and are disordered in hSET.

Stage specific expression of PfNapL-GFP chimeras. a, Western blot of transgenic cell lines expressing GFP-tagged versions of NapL with antibodies against GFP. Expression of PfHsp70 was detected to ensure similar loading. The localization of PfNapL wild type-GFP (b), PfNapL L46A-GFP (c), and PfNapL Q50A (d) in different stages of the erythrocytic life cycle of P. falciparum is shown. The first column of each panel shows bright field pictures, followed by a nuclear DAPI stain, GFP fluorescence, an overlay of the nuclear and GFP localization, and (in the last column) overlay of bright field with DAPI and GFP.

a, orthogonal views of the surface representation of hSET, yNAP-1, and PfNapL dimers with important residues from the mutagenesis data of hSET colored green and red. b, histone-binding activities of PfNapL wild type and mutants (D192A, H227A, T230A, E260A, K266A, D233A, and Y259A). Shown is binding with histone tetramer (i), histone octamer (ii), H3 (iii), H4 (iv), H2A (v), and H2B (vi).

Comparison of PfNapL dimer with the Asf1-histone complex (Protein Data Bank code 2HUE). a, superimposition of the β subdomain of PfNapL (purple ribbon) and Asf1-histone complex wherein Asf1, H3, and H4 are colored light gray, pink, and brown, respectively. The conserved residues of PfNapL are shown in a red ball-and-stick representation. The interacting residues of H3 and H4 are shown in a yellow and green ball-and-stick representation. b, close view of the proposed interaction. c, surface representation of PfNapL with predicted histone-binding residues colored red. d, histone-binding activity of PfNapL wild type and mutants (I136A and I147A) with H3-H4 tetramer.

The overall structure of PfNapL. a, domain diagram for the NAP/SET family of proteins. Histone chaperone proteins comprise a central domain and low complexity N and C termini. The inserted accessory domain in yNAP-1 (yellow) is highlighted, which is missing both in PfNapL and hSET (set to scale). The brown bar is not set to scale, since the N and C termini are variable. b, structure of the PfNapL dimer generated using 2-fold crystallographic symmetry operation. Each monomer of PfNapL contains a domain I composed of dimerization helix α2 and domain II composed of a β subdomain (four antiparallel β strands) and α-helices on the other side. c, structure-based sequence alignment of PfNapL, yNAP-1, and hSET. The structures share high structural homology, except for an additional accessory domain between domain I and domain II in yNAP-1 (foreground colored yellow). Residues contributing to dimer formation are colored green. Conserved hydrophobic motifs, predicted NES, and predicted NLS in PfNapL are underlined in purple and blue, respectively. Conserved residues from the hSET mutagenesis data are colored red and blue. d, sequence alignment of PfNapL and other eukaryotic members of the NAP family: yNAP-1 (Saccharomyces cerevisiae), xNAP-1 (Xenopus laevis), dNAP-1 (Drosophila melanogaster), and hNAP-1 (Homo sapiens). The accessory domain in yNAP-1 is foreground-colored yellow. The identical/conserved surface residues and conserved hydrophobic motifs are colored pink and purple, respectively. e, phylogenetic tree of NAPs from various species showing greater evolutionary distance of malaria parasite NAPs from homologs in yeast and humans (indicated by red arrows). f, model for in vitro relay of histones by P. falciparum nucleosome assembly proteins. This model was reproduced from Ref. 14, showing distinct roles for PfNapL (in cytoplasm as a histone carrier) and PfNapS (in nucleus capable of histone deposition). PfNapL (L) interacts with histones and can deliver histones to PfNapS (S; phosphorylated or unphosphorylated) readily. PfNapL upon phosphorylation (L^p) binds 3-fold better to the histones when compared with the unphosphorylated form of PfNapL. Phospho-PfNapL may deliver its histone cargo PfNapS. The latter takes over histones from Phospho-PfNapL-histone complexes and shuttles the histones into nucleus.

NES and NLS motifs in yNAP-1 and PfNapL. a, surface representation of yNAP-1. NES, accessory domain, and NLS are colored red, yellow, and magenta, respectively. b, surface representation of PfNapL. Predicted NES is colored red. The predicted NLS is indicated. c, sequence alignment of NES and NLS in yNAP-1 and PfNapL. The key residues are colored green. Superimposition of NES in yNAP-1 and PfNapL is shown with key residues shown in green as ball-and-stick representations.

Electrostatic potential distribution of the PfNapL dimer. The convex region of the dimerization helix α2 contains acidic residues scattered on the surface in an alternate manner. Domain II of PfNapL forms a cavity consisting of mostly acidic residues that are conserved with yNAP-1 and hSET.

Two views of the surface representation of PfNapL and yNAP-1 dimers. Corresponding residues from the mutagenesis data of hSET are colored green and red. The identical/conserved surface residues within the NAP family are colored yellow.