PDBsum entry: 1yqa

DNA binding protein

PDB id: 1yqa

Contents

Protein chain

87 a.a. *

* Residue conservation analysis

PDB id:

1yqa

Name:

DNA binding protein

Title:

Engineering the structural stability and functional properties of the gi domain into the intrinsically unfolded gii domain of the yeast linker histone hho1p

Structure:

Histone h1. Chain: a. Fragment: gii loop mutant (gii-l), residues 171-258. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: hho1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

NMR struc:

10 models

Authors:

A.Sanderson,K.Stott,T.J.Stevens,J.O.Thomas

Key ref:

A.Sanderson et al. (2005). Engineering the structural stability and functional properties of the GI domain into the intrinsically unfolded GII domain of the yeast linker histone Hho1p. J Mol Biol, 349, 608-620. PubMed id: 15878177 DOI: 10.1016/j.jmb.2005.03.085

Date:

01-Feb-05 Release date: 24-May-05

Protein chain

P53551  (H1_YEAST) -  Histone H1

Seq: 258 a.a.

Struc: 87 a.a.*

* PDB and UniProt seqs differ at 7 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Cellular component: nucleus (2 terms)
• Biological process: nucleosome assembly (1 term)
• Biochemical function: DNA binding (1 term)

DOI no: 10.1016/j.jmb.2005.03.085

J Mol Biol 349:608-620 (2005)

PubMed id: 15878177

Engineering the structural stability and functional properties of the GI domain into the intrinsically unfolded GII domain of the yeast linker histone Hho1p.

A.Sanderson, K.Stott, T.J.Stevens, J.O.Thomas.

ABSTRACT

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.

Selected figure(s)

Figure 7.
Figure 7. (a) Ensemble of 25 energy-minimised conformations of GII-L and ribbon diagram of the lowest energy structure (helices labelled). For the ensemble, ordered regions of the backbone (r.m.s.d. <0.4 Å) are illustrated in black, flexible loops in blue and flexible termini in red. (b) Ribbon diagrams of GII and GI. Asterisk indicates the kink in GI (see the text). (c) Overlays of the lowest energy structures of GII-L (colours as in (a)) with GII and GI (grey). This Figure was generated using MOLMOL[28] and ProFit (Martin, A.C.R.; http://www.bioinf.org.uk/software/profit/) for the ensemble and MolScript v2.1.2[29] and Raster3D[30] for the ribbon diagrams.

Figure 8.
Figure 8. (a) Strip plots from 3D NOESY-¹⁵N-HSQC spectra showing some of the medium-range NOEs defining helix IIa in GI and GII-L (those from the H^N of Gly81 and Gly214, respectively); the i->i+2 and i->i+3 NOEs for the corresponding residue in GII (Lys215) are absent from the GII spectrum. The NOEs for GII-L are shown schematically in (b) (MolScript v2.1.2[29] and Raster3D[30]). The red asterisks in (a) indicate an additional long-range NOE present in both GI and GII-L, from Gly81 to Phe86 and from Gly214 to Phe219, respectively, due to the tight packing of helix IIa against the hydrophobic core of the protein. This is also absent in GII. (c) Strip plots from 3D NOESY-¹³C-HSQC spectra showing long-range NOEs between the methyl groups of Val80 (GI), Val 213 (GII-L) and Leu214 (GII) and aromatic residues in the core of the protein; these long-range contacts are preserved in GII, despite the increased flexibility shown by residues in the loop.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 349, 608-620) copyright 2005.

Figures were selected by the author.

Author's comment

Abstract
Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA (approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site (“site II”) on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two “missing” site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.