PDBsum entry 1h0x

DNA binding

PDB id: 1h0x

Contents

Protein chains

89 a.a. *

Waters ×86

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure of alba: an archaeal chromatin protein modulated by acetylation.

Authors

B.N.Wardleworth, R.J.Russell, S.D.Bell, G.L.Taylor, M.F.White.

Ref.

EMBO J, 2002, 21, 4654-4662. [DOI no: 10.1093/emboj/cdf465]

PubMed id

12198167

Abstract

Eukaryotic DNA is packaged into nucleosomes that regulate the accessibility of the genome to replication, transcription and repair factors. Chromatin accessibility is controlled by histone modifications including acetylation and methylation. Archaea possess eukary otic-like machineries for DNA replication, transcription and information processing. The conserved archaeal DNA binding protein Alba (formerly Sso10b) interacts with the silencing protein Sir2, which regulates Alba's DNA binding affinity by deacetylation of a lysine residue. We present the crystal structure of Alba from Sulfolobus solfataricus at 2.6 A resolution (PDB code 1h0x). The fold is reminiscent of the N-terminal DNA binding domain of DNase I and the C-terminal domain of initiation factor IF3. The Alba dimer has two extended beta-hairpins flanking a central body containing the acetylated lysine, Lys16, suggesting three main points of contact with the DNA. Fluorescence, calorimetry and electrophoresis data suggest a final binding stoichiometry of approximately 5 bp DNA per Alba dimer. We present a model for the Alba-DNA interaction consistent with the available structural, biophysical and electron microscopy data.

Figure 2.
Figure 2 The Alba dimer. (A) Stereo views of the dimer coloured by B-factor (dark blue to deep red representing a span of B-factor from 30 to 100 Å^2). The lower view is related to the upper view by a 90° rotation around a vertical axis. The strands and helices of one monomer are labelled as in Figure 1. The N- and C-termini are highlighted by blue and red spheres, respectively. Lysines 16 and 17 are also shown. (B) Orthogonal views of the dimer showing the location of exposed residues conserved across the Archaea: A (Gly15, Lys17, Pro18, Asn21, Tyr22), B (Lys40, Arg42, Glu91) and C (Phe60). (C) Stereo view of the Alba dimer coloured by electrostatic potential. The groove formed between the two loops containing Lys16 and Lys17 is apparent. Figures 2, 3 and 5 were drawn with BOBSCRIPT (Esnouf, 1997) and GL_RENDER (L.Esser and J.Deisenhofer, unpublished).

Figure 3.
Figure 3 Comparison of DNase I with Alba. The N-terminal domain, residues 1−86, of DNase I is coloured magenta, in complex with a nicked DNA octamer (PDB code 2DNJ), and showing the -hairpin that interacts with the DNA minor groove. An Alba monomer is superimposed in yellow, revealing the more extensive -hairpin of Alba, and suggesting that the orientation of the DNA will be different in the Alba−DNA complex. The phosphorus atoms of the DNA are coloured green, and the side-chains of lysines 16 and 17 of Alba are shown.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 4654-4662) copyright 2002.

Secondary reference #1

Title

The interaction of alba, A conserved archaeal chromatin protein, With sir2 and its regulation by acetylation.

Authors

S.D.Bell, C.H.Botting, B.N.Wardleworth, S.P.Jackson, M.F.White.

Ref.

Science, 2002, 296, 148-151. [DOI no: 10.1126/science.1070506]

PubMed id

11935028

Figure 3.
Fig. 3. DNA binding affinity of Alba is modulated by acetylation. Recombinant Alba binds a 50-oligomer DNA duplex in a highly cooperative manner with an apparent dissociation constant of 33 nM (green), whereas the native acetylated protein (blue) also binds cooperatively, but more weakly (it has 1/30th the affinity). Mutants of recombinant Alba in which Lys16 or Lys17 have been changed to alanines (black) show intermediate binding affinities, whereas changes to glutamate (red) result in binding affinities similar to the acetylated protein. Binding isotherms were determined in triplicate, with standard errors indicated for each point.

Figure 4.
Fig. 4. ssSir2 mediates transcriptional silencing in vitro. (A) Transcription assays were performed on a template containing the T6 promoter of SSV1. Assay conditions were as described (13) (B) Transcription reactions were assembled with recombinant, nonacetylated (r), or acetylated (Ac) Alba. (C) Transcription reactions were assembled containing recombinant Alba with the indicated point mutations. (D) Transcription assays programmed with a plasmid containing the T6 promoter, supplemented with 1 µg of ssSir2 and/or 200 µM of NAD as indicated. (E) Upper panel: Transcription assays containing 200 µM of NAD were assembled on the T6 promoter template and incubated with Ac Alba in the presence of 1 µg of either ssSir2 or ssSir2 H116Y for 20 min at 65°C before the initiation of transcription by addition of NTPs to 200 µM. Lower panel: Transcription assays were assembled on the T6 promoter in the presence or absence of 2.5 µg of acetylated Alba. Reactions were supplemented with NAD to 200 µM and/or ssSir2 [wild type (wt) or H116Y (H-Y)]. Reactions were preincubated as above.

The above figures are reproduced from the cited reference with permission from the AAAs

Secondary reference #2

Title

Preliminary crystallographic studies of the double-Stranded DNA-Binding protein sso10b from sulfolobus solfataricus.

Authors

B.N.Wardleworth, R.J.Russell, M.F.White, G.L.Taylor.

Ref.

Acta Crystallogr D Biol Crystallogr, 2001, 57, 1893-1894. [DOI no: 10.1107/S0907444901015517]

PubMed id

11717508

Figure 1.
Figure 1 A picture of a crystal of Sso10b which is 1 mm in the longest dimension.

The above figure is reproduced from the cited reference with permission from the IUCr