pubmed.ncbi.nlm.nih.gov

The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer - PubMed

  • ️Sat Jan 01 2000

The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer

S V Brasher et al. EMBO J. 2000.

Abstract

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1beta protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.

PubMed Disclaimer

Figures

None

Fig. 1. Sequences of the chromo domains (A) and the shadow chromo domains (B) from different HP1 proteins, numbered so that they correspond to MOD1. Secondary structure elements observed in MOD1 are shown above the alignments; cylinders represent 310 (3–10) or α–helices (α1 and α2), arrows represent β–strands and circles indicate β–bulges. For each domain the residues that make up the hydrophobic core of a ‘subunit’ are shaded in yellow and other residues considered important for the structure are shown in green (Gly and Pro). Charged residues in the chromo domain, which are replaced by hydrophobic residues in the shadow chromo domain, are coloured blue (basic) and red (acidic). The red boxes enclose the structured parts of the proteins. Residues that form the dimer interface in the shadow chromo domain are boxed and shaded in grey. Mutations described in this paper are indicated below the alignment. The proteins are, from the top, mouse MOD1/human HP1β (residues 1–81 and 103–185), mouse HP1γ (1–80 and 97–173), human HP1γ (1–80 and 97–173), human HP1α (1–80 and 106–191), mouse HP1α (1–80 and 106–191), Drosophila melanogaster HP1 (1–84 and 132–206), Drosophila virilis HP1 (1–84 and 139–213) and Schizosaccharomyces pombe SWI6 (59–145 and 252–328).

None

Fig. 2. Backbone 15N T2 relaxation times for full-length MOD1 (black), free N–terminal domain and free C–terminal domain (both white). Amide groups in full-length MOD1 were assigned (where possible) by comparing spectra of the full-length protein with those of the individual domains. The T2s were calculated by non-linear least-squares fitting (Broadhurst et al., 1995).

None

Fig. 3. The structure of the shadow chromo domain dimer from MOD1. The backbone r.m.s.d. for the structure is 0.63 Å over the monomer and 0.81 Å over the dimer. (A) A stereo plot of the backbone traces of the ensemble of 16 calculated structures; the two monomers are depicted in red and blue. (B) A cartoon representation of the shadow chromo domain dimer (again red and blue) with the chromo domain from MOD1 (yellow) for comparison. (C) A close up stereo view of the inter-monomer interface with the side chains of interfacial residues shown; key residues are labelled. These plots were produced using MOLSCRIPT and Raster3D (Kraulis, 1991; Merritt and Bacon, 1997).

None

Fig. 4. MOD1C mutations disrupting the dimer structure. Wild-type MOD1C and the mutants were expressed as His–tagged fusions in E.coli, purified with Ni–NTA spin columns (Qiagen) and loaded directly onto a Superdex S75 gel–filtration column (24 ml bed volume) to assess the size of the proteins. Gel-filtration elution profiles are presented for wild-type MOD1C (wt) and the mutants W170A, W170E, Y164L, Y164E, I161E and I161A. The arrow indicates aggregates that eluted in the void volume of the column.

None

Fig. 5. Titration of MOD1C with the CAF1 MIR shows that one CAF1 peptide binds to one MOD1C dimer. The figure shows traces (A280) from gel filtration on a Superdex S75 column (2.4 ml) of MOD1C only (a) and different mixtures containing MOD1C dimer and CAF MIR in the ratios 1:0.33 (b), 1:0.8 (c), 1:1 (d), 1:2 (e) and 1:4 (f). The purity of the MOD1C and CAF MIR samples was checked by SDS–PAGE prior to mixing the proteins in the appropriate ratios. The arrows mark the positions at which free MOD1C, free CAF MIR and their complex eluted from the column. A small peak was observed eluting in the void volume of the column, suggesting that a small amount of high-molecular-weight aggregates was present in the mixtures. (Note that CAF MIR absorbs less strongly than MOD1C at 280 nm.)

None

Fig. 6. Mapping of the MIR peptide binding site on MOD1C. The molecular surface of the MOD1C dimer is shown (A), together with a cartoon of the structure (B). (The view shown is related to that in Figure 3 by a 90° rotation about the vertical axis.) Residues for which there are no data are in white, unperturbed residues are in light blue, residues for which there are two cross peaks are in blue, residues for which only one highly perturbed cross peak could be found are in magenta and the most highly perturbed residues are in red. The position of the Trp170 side chain in the binding site is shown in yellow (see the text for details). The surface plot was made using GRASP (Nicholls et al., 1991).

None

Fig. 7. (A) The MIR regions of TIF1β and CAF1 compete for binding to MOD1C. GST–TIF MIR was immobilized on glutathione–agarose beads and mixed with recombinant MOD1C. After extensive washing in buffer A150 (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol and 0.05% NP–40) the proteins remaining on the beads were separated using SDS–PAGE. The interaction between MOD1C and GST–TIF MIR was investigated in the presence of increasing amounts of a CAF1 13mer peptide containing the conserved MOD1C-binding motif. The input lanes (I) represent 20% of the amount of MOD1C present in the assay mixture, while the bound lanes (B) represent the total amount of MOD1C that remained bound to GST–TIF MIR following washing. The amounts of MOD1C and GST–TIF MIR were kept constant in each assay. (B and C) MOD1C mutants do not bind to the CAF1 MIR and TIF MIR peptides. Wild-type and mutant MOD1C proteins were in vitro translated using the TNT T7 Quick Coupled Transcription/Translation kit (Promega). Similar amounts of each 35S-labelled MOD1C protein were incubated with recombinant GST–CAF MIR (B) and GST–TIF MIR (C). After extensive washing, the proteins remaining on the beads were separated by SDS–PAGE, Coomassie Blue stained and detected by autoradiography. Auto- radiographs of the gel are presented in the figure. Lanes labelled I contain the equivalent of 20% of the input proteins. Lanes labelled B show proteins eluted from the glutathione–agarose beads following washing. Parts of the Coomassie-stained gels showing the amount of GST fusions or of GST (lanes 13 and 14) in the binding assays are presented in the bottom panels.

None

Fig. 7. (A) The MIR regions of TIF1β and CAF1 compete for binding to MOD1C. GST–TIF MIR was immobilized on glutathione–agarose beads and mixed with recombinant MOD1C. After extensive washing in buffer A150 (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol and 0.05% NP–40) the proteins remaining on the beads were separated using SDS–PAGE. The interaction between MOD1C and GST–TIF MIR was investigated in the presence of increasing amounts of a CAF1 13mer peptide containing the conserved MOD1C-binding motif. The input lanes (I) represent 20% of the amount of MOD1C present in the assay mixture, while the bound lanes (B) represent the total amount of MOD1C that remained bound to GST–TIF MIR following washing. The amounts of MOD1C and GST–TIF MIR were kept constant in each assay. (B and C) MOD1C mutants do not bind to the CAF1 MIR and TIF MIR peptides. Wild-type and mutant MOD1C proteins were in vitro translated using the TNT T7 Quick Coupled Transcription/Translation kit (Promega). Similar amounts of each 35S-labelled MOD1C protein were incubated with recombinant GST–CAF MIR (B) and GST–TIF MIR (C). After extensive washing, the proteins remaining on the beads were separated by SDS–PAGE, Coomassie Blue stained and detected by autoradiography. Auto- radiographs of the gel are presented in the figure. Lanes labelled I contain the equivalent of 20% of the input proteins. Lanes labelled B show proteins eluted from the glutathione–agarose beads following washing. Parts of the Coomassie-stained gels showing the amount of GST fusions or of GST (lanes 13 and 14) in the binding assays are presented in the bottom panels.

None

Fig. 7. (A) The MIR regions of TIF1β and CAF1 compete for binding to MOD1C. GST–TIF MIR was immobilized on glutathione–agarose beads and mixed with recombinant MOD1C. After extensive washing in buffer A150 (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol and 0.05% NP–40) the proteins remaining on the beads were separated using SDS–PAGE. The interaction between MOD1C and GST–TIF MIR was investigated in the presence of increasing amounts of a CAF1 13mer peptide containing the conserved MOD1C-binding motif. The input lanes (I) represent 20% of the amount of MOD1C present in the assay mixture, while the bound lanes (B) represent the total amount of MOD1C that remained bound to GST–TIF MIR following washing. The amounts of MOD1C and GST–TIF MIR were kept constant in each assay. (B and C) MOD1C mutants do not bind to the CAF1 MIR and TIF MIR peptides. Wild-type and mutant MOD1C proteins were in vitro translated using the TNT T7 Quick Coupled Transcription/Translation kit (Promega). Similar amounts of each 35S-labelled MOD1C protein were incubated with recombinant GST–CAF MIR (B) and GST–TIF MIR (C). After extensive washing, the proteins remaining on the beads were separated by SDS–PAGE, Coomassie Blue stained and detected by autoradiography. Auto- radiographs of the gel are presented in the figure. Lanes labelled I contain the equivalent of 20% of the input proteins. Lanes labelled B show proteins eluted from the glutathione–agarose beads following washing. Parts of the Coomassie-stained gels showing the amount of GST fusions or of GST (lanes 13 and 14) in the binding assays are presented in the bottom panels.

Similar articles

Cited by

References

    1. Aagaard L. et al. (1999)Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J., 18, 1923–1938. - PMC - PubMed
    1. Aasland R. and Stewart, A.F. (1995) The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1. Nucleic Acids Res., 23, 3168–3174. - PMC - PubMed
    1. Ball L.J. et al. (1997)Structure of the chromatin binding (chromo) domain from mouse modifier protein 1. EMBO J., 16, 2473–2481. - PMC - PubMed
    1. Broadhurst R.W., Hardman, C.H., Thomas, J.O. and Laue, E.D. (1995) Backbone dynamics of the A-domain of HMG1 as studied by 15N NMR spectroscopy. Biochemistry, 34, 16608–16617. - PubMed
    1. Brunger A.T. et al. (1998)Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D, 54, 905–921. - PubMed

Publication types

MeSH terms

Substances