PDBsum entry 2z2r

Chaperone

PDB id: 2z2r

Contents

Protein chains

275 a.a. *

* Residue conservation analysis

PDB id:

2z2r

Name:

Chaperone

Title:

Nucleosome assembly proteins i (nap-1, 74-365)

Structure:

Nucleosome assembly protein. Chain: a, b. Fragment: residues 74-365. Synonym: nap1. Engineered: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: nap1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

3.20Å R-factor: 0.260 R-free: 0.300

Authors:

Y.J.Park,K.Luger

Key ref:

Y.J.Park et al. (2008). A beta-hairpin comprising the nuclear localization sequence sustains the self-associated states of nucleosome assembly protein 1. J Mol Biol, 375, 1076-1085. PubMed id: 18068721 DOI: 10.1016/j.jmb.2007.11.031

Date:

25-May-07 Release date: 11-Mar-08

Protein chains

P25293  (NAP1_YEAST) -  Nucleosome assembly protein from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 417 a.a.

Struc: 275 a.a.

DOI no: 10.1016/j.jmb.2007.11.031

J Mol Biol 375:1076-1085 (2008)

PubMed id: 18068721

A beta-hairpin comprising the nuclear localization sequence sustains the self-associated states of nucleosome assembly protein 1.

Y.J.Park, S.J.McBryant, K.Luger.

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.

Selected figure(s)

Figure 1.
Fig. 1. Overall structures of full-length NAP1[(1–417)] and NAP1[(74–365)]. (a) The dimer structure of yNAP1[(74–365)] is shown as a ribbon diagram. Subdomains A, B, C, and D are shown in blue, yellow, green, and red, respectively (see Ref. 16 for details). Helices (α1–α8) and strands (β1–β6) are indicated. The structure of full-length yNAP1[(1–417)] (PDB access code 2ayu) is superimposed (gray). (b) Detailed view of the β-hairpin of yNAP1[(74–365)], including 3.2-Å electron density contoured at 2σ.

Figure 5.
Fig. 5. The in vitro activities of yNAP1 remain unchanged upon disruption of the β-hairpin. (a) Histone binding was analyzed by GST–NAP1 pull-down assays. A total of 0.25 nmol of purified full-length GST–NAP1(wild type), GST–NAP1(R290P), GST–NAP1(K305P), or GST alone was incubated with 100 pmol of H2A–H2B dimer. Bound proteins were analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue. Note that recombinant X. laevis H2A and H2B run in the exact same position under these conditions and that weak nonspecific interactions between H2A–H2B and GST were observed at the lowest salt conditions only. (b) Nucleosome assembly activity of wild-type and mutant NAP1. Mononucleosome assembly reactions were carried out by incubating 0.1 μM DNA with increasing amounts of histone octamer (0.1, 0.2, 0.3, and 0.4 μM), in either the absence or the presence of NAP1. Lanes 1, 6, 11, and 16, DNA alone; lanes 2–5, without NAP1; lanes 7–10, wild-type NAP1; lanes 12–15, NAP1(R290P); lanes 17–20, NAP1(K305P). The formation of mononucleosomes was analyzed by native PAGE (5% polyacrylamide, 0.2× Tris-borate-EDTA buffer), stained with SYBR Gold. The gel on the left (lanes 1–10) was stained somewhat shorter than the gel on the right, explaining the differences in DNA and nucleosome intensity. (c) yNAP1-induced H2A–H2B dimer dissociation from the nucleosome was analyzed by electrophoretic mobility shift assay. Fluorescently labeled nucleosomes were reconstituted with recombinant X. laevis histones using CPM-labeled DNA (DNA*, lanes 1–8) or H2A–H2B dimer (H2B*, lanes 9–12). Gels were viewed on a transilluminator (365 nm) without staining (the asterisk indicates fluorescently labeled nucleosomal component). The labeled nucleosomes were incubated without yNAP1 (lanes 1, 5, and 9), with wild-type NAP1 (lane 2), with NAP1(R290P) (lane 3), with NAP1(K305P) (lane 4), with wild-type GST–NAP1 (lanes 6 and 10), with GST–NAP1(R290P) (lanes 7 and 11), or with GST–NAP1(K305P) (lanes 8 and 12). Samples were incubated at 4 °C for 10 h and analyzed by 5% native PAGE. The nucleosome species N1 (centrally positioned octamer), S1 (nucleosome in which the histone octamer had shifted by 10 base pairs on the DNA), and S3 (tetrasome) are indicated (also see Ref. 11). GST–yNAP1/H2A–H2B dimer complexes (GST–S2) are shown as NCPs (H2B*). Note that the presence of more than one band of GST–S2 is likely due to the frequently described dimerization of GST.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2008, 375, 1076-1085) copyright 2008.

Figures were selected by an automated process.