Entamoeba histolytica NAP1 is a versatile histone chaperone for both H2A/H2B and H3/H4.

Abstract

Nucleosome Assembly Protein 1 (NAP1) is an evolutionarily conserved histone chaperone across eukaryotes, but the extent of functional flexibility in lineages with atypical chromatin organisation remains incompletely understood. Here, we report a comprehensive biochemical and biophysical characterisation of the NAP1 homolog from Entamoeba histolytica (EhNAP1). Despite sharing only ~20-30% sequence identity to the canonical opisthokont homologs, EhNAP1 retains a conserved homo-dimeric, largely α-helical scaffold. Unlike human and yeast NAP1, which generally favour H2A/H2B, EhNAP1 engages both histone assemblies with similar binding strengths (KD ~ 1.7-2.1 μM). EhNAP1 dimer forms a 1: 1 complex with H2A/H2B dimer and a 2: 1 complex with H3/H4 tetramer, as determined by sedimentation velocity AUC and supported by ITC. Thermodynamic analyses show that binding of both substrate types is driven by entropy, consistent with a flexible and adaptable interface for interaction. Sequence analysis revealed differences in the distribution of acidic and basic residues across histone-binding regions compared with other NAP1 homologs. Functionally, full-length EhNAP1 promotes nucleosome assembly in vitro but does not associate with pre-assembled nucleosome core particles. The C-terminal tail is identified as a key determinant for stable H2A/H2B binding. Collectively, these findings demonstrate that EhNAP1 retains the conserved NAP1 chaperone scaffold while lacking the strict substrate preference characteristic of opisthokont homologs. We propose that EhNAP1 functions as a generalist histone chaperone, representing an evolutionary adaptation to chromatin assembly in an early-diverging eukaryote with a streamlined regulatory landscape.