A β-Hairpin Comprising the Nuclear Localization Sequence Sustains the Self-associated States of Nucleosome Assembly Protein 1

Abstract

The histone chaperone nucleosome assembly protein 1 (NAP1) is implicated in histone shuttling as well as nucleosome assembly and disassembly. Under physiological conditions, NAP1 dimers exist in a mixture of various high-molecular-weight oligomers whose size may be regulated by the cell cycle-dependent concentration of NAP1. Both the functional and structural significance of the observed oligomers are unknown. We have resolved the molecular mechanism by which yeast NAP1 (yNAP1) dimers oligomerize by applying x-ray crystallographic, hydrodynamic, and functional approaches. We found that an extended beta-hairpin that protrudes from the compact core of the yNAP1 dimer forms a stable beta-sheet with beta-hairpins of neighboring yNAP1 dimers. Disruption of the beta-hairpin (whose sequence is conserved among NAP1 proteins in various species) by the replacement of one or more amino acids with proline results in complete loss of yNAP1 dimer oligomerization. The in vitro functions of yNAP1 remain unaffected by the mutations. We have thus identified a conserved structural feature of NAP1 whose function, in addition to presenting the nuclear localization sequence, appears to be the formation of higher-order oligomers.

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

“…This core domain of yNAP1 (yNap1c) is sufficient to bind the H2A–H2B dimer in solution as well as to assemble chromatin from DNA and histone components (Fujii‐Nakata et al , 1992; Park et al , 2005). The structure was determined by molecular replacement with the individual structures of the yNap1c homodimer and the histone H2A–H2B heterodimer (Fig 2B and C) (Park et al , 2008). Molecular replacement without H2A–H2B unambiguously showed the presence of the missing histones with an overall fold that was clearly visible (Figs 2 and EV1A and B).…”

“…Oligomerization of various Nap1 orthologs occurs in vivo and under physiological conditions in vitro (Ishimi et al , 1983, 1984; Fujii‐Nakata et al , 1992; Chang et al , 1997; Mosammaparast et al , 2002; McBryant & Peersen, 2004; Toth et al , 2005; Park et al , 2008; Noda et al , 2011; Newman et al , 2012). The biological role of Nap1 oligomerization has so far remained unclear.…”

“…Depending on the method of analysis, the ionic strength, and concentration used, different oligomerization states of Nap1 dimers have been reported. In size‐exclusion chromatography, purified yNap1 alone or in complex with H2A–H2B elutes as a broad peak indicative of a heterogeneous mixtures of various oligomeric species (Mosammaparast et al , 2002; Park et al , 2008). The crystal structure of yNap1c in the absence of histones revealed an extended β5–β6 hairpin that is involved in crystal packing (Park et al , 2008).…”

“…In size‐exclusion chromatography, purified yNap1 alone or in complex with H2A–H2B elutes as a broad peak indicative of a heterogeneous mixtures of various oligomeric species (Mosammaparast et al , 2002; Park et al , 2008). The crystal structure of yNap1c in the absence of histones revealed an extended β5–β6 hairpin that is involved in crystal packing (Park et al , 2008). The same packing interface is not available in crystals of full‐length yNap1, but mutation of this packing interface reduces yNap1 oligomerization in vitro (Park et al , 2008).…”

“…The crystal structure of yNap1c in the absence of histones revealed an extended β5–β6 hairpin that is involved in crystal packing (Park et al , 2008). The same packing interface is not available in crystals of full‐length yNap1, but mutation of this packing interface reduces yNap1 oligomerization in vitro (Park et al , 2008). In the presence of histones, yNap1 shows similar propensities to oligomerize (Toth et al , 2005; Noda et al , 2011).…”