Conformational Change in the Chromatin Remodelling Protein MENT

et al. (2009) Conformational Change in the Chromatin Remodelling Protein MENT. PLoS ONE 4(3): e4727. doi:10.1371/journal.pone.0004727 Conformational Change in the Chromatin Remodelling Protein MENT Poh Chee Ong 0 Sarah J. Golding 0 Mary C. Pearce 0 James A. Irving 0 Sergei A. Grigoryev 0 Debbie 0 Pike 0 Christopher G. Langendorf 0 Tanya A. Bashtannyk-Puhalovich 0 Stephen P. Bottomley 0 James C. 0 Whisstock 0 Robert N. Pike 0 Sheena McGowan 0 Adam Yuan, Temasek Life Sciences Laboratory, Singapore 0 1 Department of Biochemistry and Molecular Biology, Monash University , Clayton, Victoria , Australia , 2 Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Milton S. Hershey Medical Center , Hershey, Pennsylvania , United States of America 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. - Funding: We thank the National Health and Medical Research Council (NHMRC) of Australia and the Australian Research Council (ARC) for generous support. JCW is a ARC Federation Fellow. SPB is a NHMRC Senior Research Fellow. JAI is a NHMRC CJ Martin Postdoctoral Research Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. " These authors also contributed equally to this work. The Myeloid and Erythroid Nuclear Termination stage specific protein, MENT, is a member of the serpin superfamily [1] and is capable of inhibiting the papain-like cysteine proteases, cathepsins V and L [24]. Inhibitory serpins are metastable molecules that utilise a conformational rearrangement to inhibit target enzymes: upon interaction with a protease, the solvent-exposed reactive centre loop (RCL) is cleaved, but remains covalently bound to the protease as an acyl enzyme intermediate as it inserts into the centre of the large central b-sheet (the A-sheet) to form an additional b-strand. In the final serpin-enzyme complex, the protease is trapped in a distorted, inactive conformation [5]. MENT is also a potent chromatin remodelling protein that is responsible for heterochromatin spreading and control of terminal cell differentiation in avian erythrocytes [6]. The regions of MENT primarily responsible for interaction with nucleosomal DNA is an interhelical extension termed the M-loop, and an area centred on the D- and E-helices [3]. The genetic material of eukaryotic cells is packaged in the form of chromatin: a nuclear mass composed of DNA, histones and other associated protein. Controlling the level of compaction of chromatin is essential for the regulation of all cellular processes. Several lines of evidence suggest that there may be a relationship between the inhibitory activity of MENT and its role in condensing chromatin: (1) protease inhibition by MENT contributes to chromatin rearrangements in vivo [2,7]; (2) cathepsin V, a nuclear protease implicated in the control of the transcription factor CDP/CUX, is rendered susceptible to inhibition by MENT in the presence of DNA [8]; and (3) MENT is able to form proteinprotein bridges, mediated by the RCL, that may be important for chromatin remodelling [3]. In the crystal structure of wild-type MENT, two residues of the RCL are inserted into the top of the A b-sheet. Most serpins characterised to date that exhibit such partial RCL insertion undergo co-factor mediated conformational shifts; for example antithrombin and heparin co-factor II both undergo RCL expulsion in the presence of heparin. Therefore, we have used DNA, both naked and nucleosome-bound, to investigate conformational change in MENT by measuring changes in intrinsic tryptophan fluorescence. It was found that soluble chromatin was able to induce conformational change in MENT in contrast to naked DNA that was unable to induce any change. To attempt to determine the basic structure required for a conformational change in MENT, we constructed mononucleosomes and found that this was the necessary structure required for a conformational change in the molecule. We further probed this conformational rearrangement by analysing the changes seen in key MENT binding mutants and deconvolution of the tryptophan emission spectra using single tryptophan variants of MENT. Results and Discussion MENTWT undergoes a conformational change in the presence of chromatin that is independent of the RCL sequence but dependent upon the M-loop and D-helix residues The mechanism by which nucleosome arrays are folded into higher order structures remains somewhat obscure. While the structures of individual nucleosomes and chromatin remodelling proteins have proved essential to understanding chromatin structure, traditional crystallographic approaches to higher order chromatin structure determination have been hampered by a lack of homogeneity as well as mobility in higher order chromatin structures. Thus, biophysical studies have proved central to our understanding of protein mobility during chromatin remodelling [9,10]. Conformational flexibility in serpins is well characterised and represents a crucial evolutionary advantage of their function [11]. In order to ascertain if the interaction of MENT with chromatin during condensation induced any structural changes (...truncated)