A biochemical and biophysical study of nucleosome assembly by the <i>Saccharomyces cerevisiae</i> Nucleosome Assembly Protein 1
Nucleosome Assembly Protein 1 (Nap1) is a highly conserved eukaryotic histone chaperone that preferrentially binds histones H2A and H2B in vivo and can facilitate nucleosome assembly in vitro. To gain insight into the mechanism of Nap1-mediated nucleosome assembly we performed a detailed thermodynamic analysis of the Nap1-H2AH2B complex using recombinant Saccharomyces cerevisiae Nap1 (yNap1) and H2AH2B heterodimers. We find yNap1 is predominantly a dimer in solution and has a separable bipartite structure in which residues 81-150 (domain I) mediate dimerization and residues 172-372 (domain II) form a domain for binding yH2AH2B. Isothermal titration calorimetry reveals that a yNap1 dimer binds two yH2AH2B heterodimers via principal interaction sites in the two domain IIs, which bind to the historic fold region of yH2AH2B with a Kd < 10 nM. Binding to additional yH2AH2B can occur via separate, lower affinity sites located in the acidic C-terminal tail of yNap1, which bind to the histone N-terminal tail regions with a Kd ∼ 110 nM. Previous studies have demonstrated that the acidic C-terminal tail of yNap1 is dispensable for in vitro nucleosome assembly activity, and therefore the minimum requirement for a functional Nap1-H2AH2B complex is a yNap1 dimer bound to two yH2AH2B. Analysis of the nucleosome assembly activity of full-length yNap1 and yNap1 truncation mutants suggests a concerted mechanism of H2AH2B deposition by Nap1 that rationalizes the rapid, high-fidelity assembly of nucleosomes in vitro by Nap1. The proposed mechanism of nucleosome assembly is placed in context of recent publications.