Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding

Abstract

Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic Nterminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in selfassociation, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4) 2 tetramer can simultaneously bind two Nap 2 protein homodimers.

Structural Insights into ceNAP1 Chaperoning Activity toward ceH2A-H2B

“…Our complex structure shows that only one copy of H2A-H2B heterodimer binds a headphone-like ceNAP1 homodimer at one earmuff domain (one side of the concave surface) of the ceNAP1 homodimer (Fig- ure 3B). The 1:1 molar ratio was consistent with the recent biochemical characterization of full-length ceNAP1 at 300 mM NaCl (Sarkar et al, 2019). The molar ratio of our complex structure is the same as that reported previously in the low-resolution scNAP1-H2A-H2B complex (Aguilar-Gurrieri et al, 2016).…”

“…Our SEC-MALS experiments of ceNAP1 C -xgH2A-gH2B (Figure S4A) (or ceNAP1 C -ceH2A-H2B, Figure S4B) at 200 mM NaCl showed that the average MW of the ceNAP1 C -H2A-H2B complex is larger than its theoretical MW, indicating the presence of oligomers (ceNAP1-H2A-H2B) n . These are consistent with previous SEC-MALS data of full-length ceNAP1-H2A-H2B at 150 mM NaCl (Sarkar et al, 2019). The oligomer states of ceNAP1 C -xgH2A-xgH2B were further investigated at 200 mM NaCl using AUC-SV.…”

“…Previous mass spectra and analytical ultracentrifugation sedimentation velocity (AUC-SV) experiments revealed that both hNAP1 and scNAP1 dimers form tetramers, and even higher assembly states under physiological conditions (Noda et al, 2011). Recent research on full-length ceNAP1 suggests a dimertetramer equilibrium at 150 mM NaCl (Sarkar et al, 2019). Our size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) data at 200 mM NaCl (Figure 2A) indicated a molecular weight (MW) of ceNAP1 C ranging from the dimer state (theoretical dimer MW = 69.9 kDa) to the tetramer state (theoretical tetramer MW = 139.8 kDa): the observed MW was 93.9 kDa.…”

“…Compared with NAP1 from other species, full-length ceNAP1, with only 316 residues, naturally lacks an acidic N-terminal tail, but possesses a short acidic C-terminal tail (Figure 1A). Recent research reported a crystal structure of the full-length ceNAP1 dimer at 3 Å resolution (PDB: 6N2G), and the stoichiometry of the NAP1-H2A-H2B interaction (Sarkar et al, 2019), but no complex structure with histones was presented.…”

Structure-function relationship of H2A-H2B specific plant histone chaperones

“…NAP1, initially obtained from Xenopus laevis eggs and identified as a protein that facilitates the in vitro assembly of nucleosomes, is conserved extensively across different species (Laskey et al 1978). NAP family proteins have been reported from a wide range of organisms, such as yeast (Ishimi and Kikuchi 1991), Plasmodium (Gill et al 2009), Drosophila (Ito et al 1996), Xenopus (Steer et al 2003), human (Li et al 2016), Caenorhabditis elegans (Sarkar et al 2019), soya bean (Yoon et al 1995), rice (Dong et al 2003), tobacco (Dong et al 2003), and Arabidopsis (Liu et al 2009). The representative members of NAP family include Vps75 in yeast (Berndsen et al 2008;Tang et al 2008), hsNAPIL and xlNAPIL in human and Xenopus laevis, respectively, SETα and SETβ in humans and metazoans, and NAP1 related protein 1 (NRP1) and NAP1 related protein 2 (NRP2) in A. thaliana (Zhu et al 2006).…”