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Structural studies of a four-MBT repeat protein MBTD1.

Eryilmaz J, Pan P, Amaya MF, Allali-Hassani A, Dong A, Adams-Cioaba MA, Mackenzie F, Vedadi M, Min J - PLoS ONE (2009)

Bottom Line: All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1].Please note that a web plugin is required to access this enhanced functionality.Instructions for the installation and use of the web plugin are available in Text S1.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats.

Methodology/principal findings: We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 A resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1]. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation.

Conclusions: The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage.

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Overall structure of MBTD1.There are two MBTD1 molecules in the asymmetric unit, and each molecule contains four MBT repeats (MBT1, MBT2, MBT3, MBT4), which exhibit irregular rhombus architecture.
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pone-0007274-g001: Overall structure of MBTD1.There are two MBTD1 molecules in the asymmetric unit, and each molecule contains four MBT repeats (MBT1, MBT2, MBT3, MBT4), which exhibit irregular rhombus architecture.

Mentions: MBTD1 is a four MBT repeat protein comprising 628 amino acids. It contains a FCS-type zinc finger at the N-terminus with putative regulatory function [22] and four MBT repeats at the C-terminus. To investigate the crystal structure of the four MBT repeat fragment of MBTD1 and characterize its histone binding specificity, we cloned and purified a human MBTD1 fragment composed of all four MBT repeats (residues 130–566). MBTD1130–566 crystallized in the orthorhombic space group P212121 (a = 70.31 Å, b = 100.90 Å, c = 135.30 Å) with two molecules in the asymmetric unit (Figure 1). Crystal diffraction data and refinement statistics for the MBTD1 structure are displayed in Table 1. In the MBTD1 structure, each molecule contains four MBT repeats that exhibit irregular rhomboid architecture. A narrow channel runs through the middle of the structure and is filled with water molecules. Consistent with previously reported MBT repeat structures, each MBT repeat contains an extended “arm” which packs against a globular β subunit core of the preceding repeat [1]. Structural and sequence alignment show that the β-barrel subunit core region has the highest conservation between MBTD1 and other MBT repeat proteins. The residues involved in intermolecular hydrogen bond interactions between the two molecules in the asymmetric unit are conserved throughout the whole MBT repeat family, possibly implying that MBTD1 functions as a dimer unit (Figure 2). Nonetheless, dynamic light scattering (DLS) experiments and size exclusion experiments demonstrate that MBTD1 is a monomer in solution at the working concentrations (∼5 mg/ml, data not shown). Structurally, the last three MBT repeats in MBTD1 and L3MBTL2 form three-blade propeller architecture. Superposition of the three MBT repeats of L3MBTL1 with those of MBTD1 and L3MBTL2 shows a good structural alignment of Cα positions with a root-mean-squared deviation (RMSD) of 2.345 Å and 2.168 Å, respectively. Furthermore, the MBTD1 can also be well superimposed with L3MBTL2 with a root-mean-squared deviation of 0.697 Å.


Structural studies of a four-MBT repeat protein MBTD1.

Eryilmaz J, Pan P, Amaya MF, Allali-Hassani A, Dong A, Adams-Cioaba MA, Mackenzie F, Vedadi M, Min J - PLoS ONE (2009)

Overall structure of MBTD1.There are two MBTD1 molecules in the asymmetric unit, and each molecule contains four MBT repeats (MBT1, MBT2, MBT3, MBT4), which exhibit irregular rhombus architecture.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747274&req=5

pone-0007274-g001: Overall structure of MBTD1.There are two MBTD1 molecules in the asymmetric unit, and each molecule contains four MBT repeats (MBT1, MBT2, MBT3, MBT4), which exhibit irregular rhombus architecture.
Mentions: MBTD1 is a four MBT repeat protein comprising 628 amino acids. It contains a FCS-type zinc finger at the N-terminus with putative regulatory function [22] and four MBT repeats at the C-terminus. To investigate the crystal structure of the four MBT repeat fragment of MBTD1 and characterize its histone binding specificity, we cloned and purified a human MBTD1 fragment composed of all four MBT repeats (residues 130–566). MBTD1130–566 crystallized in the orthorhombic space group P212121 (a = 70.31 Å, b = 100.90 Å, c = 135.30 Å) with two molecules in the asymmetric unit (Figure 1). Crystal diffraction data and refinement statistics for the MBTD1 structure are displayed in Table 1. In the MBTD1 structure, each molecule contains four MBT repeats that exhibit irregular rhomboid architecture. A narrow channel runs through the middle of the structure and is filled with water molecules. Consistent with previously reported MBT repeat structures, each MBT repeat contains an extended “arm” which packs against a globular β subunit core of the preceding repeat [1]. Structural and sequence alignment show that the β-barrel subunit core region has the highest conservation between MBTD1 and other MBT repeat proteins. The residues involved in intermolecular hydrogen bond interactions between the two molecules in the asymmetric unit are conserved throughout the whole MBT repeat family, possibly implying that MBTD1 functions as a dimer unit (Figure 2). Nonetheless, dynamic light scattering (DLS) experiments and size exclusion experiments demonstrate that MBTD1 is a monomer in solution at the working concentrations (∼5 mg/ml, data not shown). Structurally, the last three MBT repeats in MBTD1 and L3MBTL2 form three-blade propeller architecture. Superposition of the three MBT repeats of L3MBTL1 with those of MBTD1 and L3MBTL2 shows a good structural alignment of Cα positions with a root-mean-squared deviation (RMSD) of 2.345 Å and 2.168 Å, respectively. Furthermore, the MBTD1 can also be well superimposed with L3MBTL2 with a root-mean-squared deviation of 0.697 Å.

Bottom Line: All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1].Please note that a web plugin is required to access this enhanced functionality.Instructions for the installation and use of the web plugin are available in Text S1.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats.

Methodology/principal findings: We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 A resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1]. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation.

Conclusions: The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage.

Enhanced version: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Show MeSH
Related in: MedlinePlus