Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

Abstract

Summary The histone H2A-H2B heterodimer is an integral component of the nucleosome. The cellular localization and deposition of H2A-H2B into chromatin is regulated by numerous factors including histone chaperones such as Nucleosome Assembly Protein 1 (Nap1). We use hydrogen-deuterium exchange coupled to mass spectrometry to characterize H2A-H2B and Nap1. Unexpectedly, we find that at low ionic strength the α-helices in H2A-H2B are frequently sampling partially disordered conformations, and that binding to Nap1 reduces this conformational sampling. We identify the interaction surface between H2A-H2B and Nap1, and confirm its relevance both in vitro and in vivo. We show that two copies of H2A-H2B bound to a Nap1 homodimer form a tetramer with contacts between H2B chains similar to those in the four-helix bundle structural motif. The organization of the complex reveals that Nap1 competes with histone-DNA and inter-histone interactions observed in the nucleosome, thereby regulating the availability of histones for chromatin assembly.

Structural evidence for Nap1‐dependent H2A–H2B deposition and nucleosome assembly

“…However, in the presence of H2A–H2B, self‐assembly was more pronounced when these flexible tails were absent. In all experiments, and in agreement with our structure, we found that a single H2A–H2B dimer binds to a yNap1 dimer and we could not confirm the stoichiometry proposed by D'Arcy et al (2013). These data confirm that a single yNap1 dimer binds to a single H2A–H2B heterodimer and that this complex self‐assembles into higher‐order oligomers.…”

“…Previous low‐resolution hydrogen–deuterium exchange experiments suggested that a yNap1 dimer accommodates two copies of H2A–H2B in a tetrameric conformation (D'Arcy et al , 2013). Instead, we found that only a single H2A–H2B heterodimer can be accommodated in the binding pocket of a Nap1 dimer.…”

“…A stoichiometry of both one and two histone dimers to a single Nap1 dimer has been proposed (McBryant et al , 2003; Toth et al , 2005; Andrews et al , 2008; Newman et al , 2012; D'Arcy et al , 2013). By using hydrogen–deuterium exchange, D'Arcy et al (2013) reported that a yNap1 dimer accommodates two copies of H2A–H2B in an unconventional tetramer conformation. Such low‐resolution techniques can produce confounding results, especially with self‐assembling systems where it is difficult to determine from which interface (Nap1–Nap1, Nap1–histone, or histone–histone) protection arises.…”

“…Our structure and extensive native mass spectrometry experiments now demonstrate that only a single H2A–H2B heterodimer is accommodated in the binding pocket of a yNap1 dimer. D'Arcy et al (2013) performed extensive mutagenesis of the acidic underside of yNap1 with the goal to identify the histone binding region. Our structure now shows that the mutations identified to disrupt histone binding are not located in the histone binding interface.…”

Математическое Моделирование Образования Комплекса Белковых Молекул С Учетом Их Доменной Структуры

“…Так, в статье [1] приведен обзор раз-личных функций белка Nap1, преимущественно его уча-стие в сборке и разборке нуклеосомы, взаимодействие белка Nap1 с различными факторами ремоделирования хроматина, приведена информация о различных участках связывания белка Nap1 с другими белками. В [2] авторы использовали водно-дейтериевый обмен в сочетании с масс-спектрометрией для нахождения участков связыва-ния димера (H2A−H2B) с белком Nap1. В результате выполненной работы авторы установили, что при низкой ионной силе гистоны в димере (H2A−H2B) могут находиться в неупорядоченной конформации, однако связывание димера (H2A−H2B) с Nap1 уменьшает эту структурную неупорядоченность.…”