Crystal structure and function of human nucleoplasmin (Npm2): a histone chaperone in oocytes and embryos

Npm2 is the mammalian ortholog of nucleoplasmin (Np), a well characterized histone chaperone in Xenopus oocytes and early embryos. 1,2 Nucleoplasmin 2 expression is limited to oocytes, where it accumulates in nuclei and is excluded from nucleoli. 3 In mouse embryos, Npm2 is present until the 8-cell stage and is then down-regulated. 3 In npm2−/− mice there is a reduced cleavage of fertilized eggs to the two-cell stage, which renders these females less fertile. In addition, the architecture of cell nuclei is altered in knockout cells, as nucleoli appear fragmented and chromatin compaction is adversely affected. These defects persist in early zygotes. 3 Thus, Npm2 may function as a histone chaperone to help remodel chromatin in oocytes and early embryos, 3 but this has not been demonstrated directly. Interestingly, Npm2 does not appear to be required for sperm chromatin decondensation 3, while Np plays an important role in this process in Xenopus eggs during fertilization.2 The general importance of histone chaperones is shown by the propensity of DNA to be damaged when nucleosome assembly is compromised and conversely, free histones may also be detrimental to cells.4 In Xenopus oocytes, Np may form storage complexes with H2A-H2B dimers while N1 associates with H3-H4 tetramers. Working in concert, these chaperones may direct nucleosome assembly in the early embryo. 5–7 Npm2 and Np have an overall sequence identity of ~46%. Both chaperones contain a conserved N-terminal domain (the core), followed by a C-terminal tail that contains two acidic tracts (denoted A2 and A3), with a bipartite nuclear localization signal located between them (Figure 1A). 8 Acidic tracts in Np are disordered and play a role in binding core histones 9–11 and linker histones. 12 This includes a short acidic loop in the N-terminal domain (the A1-tract), which may interact with sperm basic proteins 13,14 and histones. 9–11 Figure 1 Domain organization, sequence alignment and monomer structure of Npm2 Crystal structures have been determined for N-terminal domains of Xenopus Np and NO38 9,11,15 Drosophila NLP 10 and human Npm1. 16 This core domain is responsible for pentamer formation and in three of the crystal structures, decamers are formed by the face-to-face association of two pentamers. In each decamer, salt bridges are formed by conserved residues in the AKDE and K-loops of opposing subunits in the two pentamers. 9,11,16 Pentamers and decamers in the Np-family may each play a role in histone binding 9–11,16, but the precise mechanism is currently being debated. 17–19 In this paper, we present the crystal structure of a human Npm2-core pentamer. We then analyzed histone binding by Npm2-core and a longer version of the chaperone that contains a large acidic tract in the C-terminal tail (Npm2-A2). These studies show that hNpm2 requires the A2-tract to bind core histones. This is due to the fact that each A1-loop in Npm2 contains only a single acidic residue, which greatly reduces its electrostatic contribution to histone binding. We also made loop exchange mutants between Npm2 and Np to test the roles of the A1- and A2-tracts. On balance, we find the A1- and A2-tracts of Np may act synergistically in histone binding. We also show that Npm2 and Np form decamers when they bind the four core histones, while H2A-H2B dimers bind to a single pentamer. We then modeled an Npm2 decamer to reveal compensatory changes in residues of the AKEE and Q-loops of this chaperone. These residues may form hydrogen bonds between opposing pentamers when chaperone decamers are formed during histone binding. Finally, we present FRET and biochemical data to support a model in which H2A-H2B dimers bind directly to nucleoplasmins, followed by the addition of H3-H4 tetramers at higher radius to form larger complexes.