Nucleoplasmin Binds Histone H2A-H2B Dimers through Its Distal Face*

Abstract

Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chromatin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B histone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here, we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers and that the amount of bound ligand depends on the overall charge (phosphorylation level) of the chaperone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information can help to understand the function of NP as a histone chaperone.

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones

“…diluting the histones from 2 M NaCl solutions, were subjected to trypsin treatment, and the resulting fragments were analyzed by MS. As controls, isolated eNP (Figure 5A; lanes 1 and 2) and H3-H4 (Figure 5A; lanes 3–5) were also digested. Data show, as previously found for H2A-H2B (21), that histones were extensively digested at low ionic strength, i.e. 0.24 M NaCl (Figure 5A, lane 4), whereas at 2 M NaCl, stable fragments were observed (Figure 5A, lane 3).…”

“…Moreover, when the H2A-H2B/H3-H4 mixture is extensively dialyzed against 0.24 M NaCl buffer, conditions that favor H3-H4 dimerization, the amount of the high MW complex is negligible (Figure 3D). The sedimentation profile of this sample resembles that of eNP/H2A-H2B complexes (21), suggesting that the chaperone interacts mainly with histone dimers generated on octamer/H3-H4 tetramer dissociation.

Figure 3.NP binds differently H3-H4 dimers and tetramers.

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“…( D ) The 2D average image of the eNP/H3-H4 1/2 complex (average of 412 particles). The 2D average images of eNP ( E ) and of the eNP/H2A-H2B complex ( F ) (21). Bar represents 200 Å in A and 100 Å in (C–F).…”

Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs

“…Because endogenous H2A is only found bound to Npm in the egg, the likely endogenous histone substrate for Prmt5-Mep50 is bound to Npm. Npm binding to histones possibly alters the conformation so that the unstructured tails of both Npm and histones may be no longer accessible for binding and methyltransferase activity (54). Furthermore, endogenous modifications on Npm, including phosphorylation and arginine methylation, substantially alter the Prmt5 activity toward histones as well.…”

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

“…The C-terminal intrinsically disordered domain contains a bipartite nuclear localization sequence, A2 and A3, and the extreme C-terminus containing positive amino acids (Dutta et al, 2001; Prado et al, 2004). Previous biochemical and electron microscope analyses revealed that the core is sufficient to bind histones, but the tail also engages in histone binding (Arnan et al, 2003; Ramos et al, 2014; Ramos et al, 2010; Taneva et al, 2009). The functional significance of the tail binding is unknown.…”