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

•Nap1 stabilizes the fold of H2A-H2B•An α-helical region of Nap1 directly binds H2B•Nap1 and DNA directly compete for H2A-H2B•Two copies of H2A-H2B bound to Nap1 can form a tetramer with contacts between H2B

SummaryThe 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 interhistone interactions observed in the nucleosome, thereby regulating the availability of histones for chromatin assembly.