Conformational change in the chromatin remodelling protein MENT

Poh Chee Ong, Sarah J. Golding, Mary C. Pearce, James A. Irving, Sergei A. Grigoryev, Debbie Pike, Christopher G. Langendorf, Tanya A. Bashtannyk-Puhalovich, Stephen P. Bottomley, James C. Whisstock, Robert N. Pike, Shenna McGowan

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Abstract

Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding 'M-loop' and D-helix of MENT, but is independent of the protease specificity determining 'reactive centre loop'. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.

Original languageEnglish (US)
Article numbere4727
JournalPloS one
Volume4
Issue number3
DOIs
StatePublished - Mar 6 2009

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Ong, P. C., Golding, S. J., Pearce, M. C., Irving, J. A., Grigoryev, S. A., Pike, D., Langendorf, C. G., Bashtannyk-Puhalovich, T. A., Bottomley, S. P., Whisstock, J. C., Pike, R. N., & McGowan, S. (2009). Conformational change in the chromatin remodelling protein MENT. PloS one, 4(3), [e4727]. https://doi.org/10.1371/journal.pone.0004727