PDBsum entry 3bip

Transcription

PDB id

3bip

Contents

			Protein chains

					444 a.a.

			Waters ×721

References listed in PDB file

Key reference

Title

Structural and functional analysis of the spt16p n-Terminal domain reveals overlapping roles of yfact subunits.

Authors

A.P.Vandemark, H.Xin, L.Mccullough, R.Rawlins, S.Bentley, A.Heroux, D.J.Stillman, C.P.Hill, T.Formosa.

Ref.

J Biol Chem, 2008, 283, 5058-5068. [DOI no: 10.1074/jbc.M708682200]

PubMed id

18089575

Abstract

yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.



	Figure 1. FIGURE 1. Structure of the Spt16 NTD. Top, the residues in each structural domain of Spt16-Pob3 are indicated (16, 23). 31 Spt16 homologs chosen to include the full spectrum of eukaryotes were aligned and found to be about 40% identical to the yeast sequence overall. The percent identity to the S. cerevisiae sequence varied for each domain as indicated. All homologs included the NTD. The C-terminal domain is broken into two regions for this calculation, a less conserved but 50% acidic region (960-1008) and a more highly conserved and neutral region (1009-1029). D indicates the dimerization interface. Middle, ribbon diagrams of the Spt16 NTD structure. The N-terminal lobe is shown in turquoise; the C-terminal lobe containing the putative binding cleft in blue, and the loop that encloses the cleft in orange. Labels indicate the secondary structure features. Bottom, secondary structures within the Spt16 NTD are aligned with the sequence. Residues that are conserved among over 70% of the 31 Spt16 homologs compared are highlighted in red. Bars below the text indicate regions targeted for site-directed mutagenesis, coded according to the severity of the synthetic defect with pob3-Q308K (Table 2).		Figure 5. FIGURE 5. Conservation correlates with the strength of synthetic defects with pob3-Q308K. Top, residues that are identical among at least 70% of the 31 Spt16 homologs aligned (see Fig. 1) are indicated in red, revealing clustering near the canonical binding/active site cleft for prolidase, methionine aminopeptidase, aminopeptidase P, and creatinase (asterisk). The full surface is shown on the left, and the loop residues 266-274 are removed in the middle panel to reveal the enclosed tunnel region. The right panel shows a view rotated 180° about the vertical axis. Bottom, severity of the synthetic defect when combined with pob3-Q308K is indicated (Table 2); severe defects are shown in red, moderate defects in orange, and mild defects in yellow. If residues were tested individually and in multiple mutations, only the score from the single mutation is used here. Otherwise, the score for the complex mutation is assigned to all residues altered. WT sequences are indicated along with the number of the first residue. The orientations are the same as in the top panels.

PROCHECK

	Headers