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Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes.

Yang C, van der Woerd MJ, Muthurajan UM, Hansen JC, Luger K - Nucleic Acids Res. (2011)

Bottom Line: We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present.MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA.In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Howard Hughes Medical Institute, Colorado State University, Fort Collins, CO 80523-1870, USA.

ABSTRACT
MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

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SAXS data show that MeCP2 interacts differently with A-147Nuc compared with W-165Nuc. (A) Normalized P(r) functions for A-Nuc147 and complexes with full length and truncated MeCP2 (78–305) show that the complex of the nucleosome and MeCP2 has approximately the same dimension as the sum of its parts. (B) Normalized P(r) functions for W-Nuc165 and complexes with full length and truncated MeCP2 show that the complex is similar in size and character to the nucleosome by itself. (C) Superposition of average envelopes reconstructed from SAXS data, with the A-Nuc147—MeCP2 complex in transparent gray and the corresponding nucleosome in blue. (D) Same as (C) for the W-Nuc165 – MeCP2 complex and W-Nuc165, respectively.
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Figure 7: SAXS data show that MeCP2 interacts differently with A-147Nuc compared with W-165Nuc. (A) Normalized P(r) functions for A-Nuc147 and complexes with full length and truncated MeCP2 (78–305) show that the complex of the nucleosome and MeCP2 has approximately the same dimension as the sum of its parts. (B) Normalized P(r) functions for W-Nuc165 and complexes with full length and truncated MeCP2 show that the complex is similar in size and character to the nucleosome by itself. (C) Superposition of average envelopes reconstructed from SAXS data, with the A-Nuc147—MeCP2 complex in transparent gray and the corresponding nucleosome in blue. (D) Same as (C) for the W-Nuc165 – MeCP2 complex and W-Nuc165, respectively.

Mentions: Analysis of the SAXS data for full length MeCP2 further documents that MeCP2 is an extended, largely disordered molecule [Table 2; compare Kratky plots in Supplementary Figure S6 for A-Nuc147, MeCP2 alone and in complex with A-Nuc147; (8)]. The extended nature is also apparent from its P(r) function, which shows many more long intra-particle distances than a globular molecule and a large maximum (linear) particle dimension, dmax (Figure 7A). Both features are indicative of an extended molecule. Upon complex formation with A-Nuc147, MeCP2 maintains a rather extended conformation, as seen in the P(r) function of the complex and in a dmax of the complex that is 50 Å larger than that obtained for nucleosome alone (Table 2). Ab initio calculated envelopes demonstrate that the basic shape of the nucleosome is maintained and display an asymmetric relatively narrow extension which we interpret to be MeCP2 (Figure 7C). Because the structure of full length MeCP2 is unknown and the protein is known to be mostly disordered (9), rigid body modeling of the complex is not feasible. A particle of A-Nuc147 in complex with a fragment of MeCP2 that only contains the first of at least three DNA binding sites [MeCP2(74–305); Figure 1] exhibits only a slightly increased dmax compared with the unbound nucleosome (Table 2). This suggests that the DBDs in this entire fragment are engaged in interactions, resulting in a global shape and size for the complex that does not differ significantly from that of the unbound nucleosome. Both of these particles represent 1:1 stoichiometric complexes between MeCP2 and the nucleosome, as verified by SEC-MALS, and consistent with the Porod volumes (Supplementary Figure S2, Table 2).Figure 7.


Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes.

Yang C, van der Woerd MJ, Muthurajan UM, Hansen JC, Luger K - Nucleic Acids Res. (2011)

SAXS data show that MeCP2 interacts differently with A-147Nuc compared with W-165Nuc. (A) Normalized P(r) functions for A-Nuc147 and complexes with full length and truncated MeCP2 (78–305) show that the complex of the nucleosome and MeCP2 has approximately the same dimension as the sum of its parts. (B) Normalized P(r) functions for W-Nuc165 and complexes with full length and truncated MeCP2 show that the complex is similar in size and character to the nucleosome by itself. (C) Superposition of average envelopes reconstructed from SAXS data, with the A-Nuc147—MeCP2 complex in transparent gray and the corresponding nucleosome in blue. (D) Same as (C) for the W-Nuc165 – MeCP2 complex and W-Nuc165, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105411&req=5

Figure 7: SAXS data show that MeCP2 interacts differently with A-147Nuc compared with W-165Nuc. (A) Normalized P(r) functions for A-Nuc147 and complexes with full length and truncated MeCP2 (78–305) show that the complex of the nucleosome and MeCP2 has approximately the same dimension as the sum of its parts. (B) Normalized P(r) functions for W-Nuc165 and complexes with full length and truncated MeCP2 show that the complex is similar in size and character to the nucleosome by itself. (C) Superposition of average envelopes reconstructed from SAXS data, with the A-Nuc147—MeCP2 complex in transparent gray and the corresponding nucleosome in blue. (D) Same as (C) for the W-Nuc165 – MeCP2 complex and W-Nuc165, respectively.
Mentions: Analysis of the SAXS data for full length MeCP2 further documents that MeCP2 is an extended, largely disordered molecule [Table 2; compare Kratky plots in Supplementary Figure S6 for A-Nuc147, MeCP2 alone and in complex with A-Nuc147; (8)]. The extended nature is also apparent from its P(r) function, which shows many more long intra-particle distances than a globular molecule and a large maximum (linear) particle dimension, dmax (Figure 7A). Both features are indicative of an extended molecule. Upon complex formation with A-Nuc147, MeCP2 maintains a rather extended conformation, as seen in the P(r) function of the complex and in a dmax of the complex that is 50 Å larger than that obtained for nucleosome alone (Table 2). Ab initio calculated envelopes demonstrate that the basic shape of the nucleosome is maintained and display an asymmetric relatively narrow extension which we interpret to be MeCP2 (Figure 7C). Because the structure of full length MeCP2 is unknown and the protein is known to be mostly disordered (9), rigid body modeling of the complex is not feasible. A particle of A-Nuc147 in complex with a fragment of MeCP2 that only contains the first of at least three DNA binding sites [MeCP2(74–305); Figure 1] exhibits only a slightly increased dmax compared with the unbound nucleosome (Table 2). This suggests that the DBDs in this entire fragment are engaged in interactions, resulting in a global shape and size for the complex that does not differ significantly from that of the unbound nucleosome. Both of these particles represent 1:1 stoichiometric complexes between MeCP2 and the nucleosome, as verified by SEC-MALS, and consistent with the Porod volumes (Supplementary Figure S2, Table 2).Figure 7.

Bottom Line: We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present.MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA.In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Howard Hughes Medical Institute, Colorado State University, Fort Collins, CO 80523-1870, USA.

ABSTRACT
MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

Show MeSH
Related in: MedlinePlus