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.
References
[1]
Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, et al. (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276: 33293–33296.
[2]
Irving JA, Shushanov SS, Pike RN, Popova EY, Bromme D, et al. (2002) Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation. J Biol Chem 277: 13192–13201.
[3]
McGowan S, Buckle AM, Irving JA, Ong PC, Bashtannyk-Puhalovich TA, et al. (2006) X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation. Embo J 25: 3144–3155.
[4]
Springhetti EM, Istomina NE, Whisstock JC, Nikitina T, Woodcock CL, et al. (2003) Role of the M-loop and reactive center loop domains in the folding and bridging of nucleosome arrays by MENT. J Biol Chem 278: 43384–43393.
[5]
Huntington JA, Read RJ, Carrell RW (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407: 923–926.
[6]
Grigoryev SA, Solovieva VO, Spirin KS, Krasheninnikov IA (1992) A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis. Exp Cell Res 198: 268–275.
[7]
Bulynko YA, Hsing LC, Mason RW, Tremethick DJ, Grigoryev SA (2006) Cathepsin L stabilizes the histone modification landscape on the Y chromosome and pericentromeric heterochromatin. Mol Cell Biol 26: 4172–4184.
[8]
Ong PC, McGowan S, Pearce MC, Irving JA, Kan WT, et al. (2007) DNA accelerates the inhibition of human cathepsin V by serpins. J Biol Chem 282: 36980–36986.
[9]
Mazumder A, Shivashankar G (2007) Gold-nanoparticle-assisted laser perturbation of chromatin assembly reveals unusual aspects of nuclear architecture within living cells. Biophys J 93: 2209–2216.
[10]
Montel F, Fontaine E, St-Jean P, Castelnovo M, Faivre-Moskalenko C (2007) Atomic force microscopy imaging of SWI/SNF action: mapping the nucleosome remodeling and sliding. Biophys J 93: 566–578.
Meagher JL, Beecham JM, Olson ST, Gettins PG (1998) Deconvolution of the fluorescence emission spectrum of human antithrombin and identification of the tryptophan residues that are responsive to heparin binding. J Biol Chem 273: 23283–23289.
[13]
Lowary PT, Widom J (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. Journal of Molecular Biology 276: 19–42.
[14]
Hasselbacher CA, Rusinova E, Waxman E, Rusinova R, Kohanski RA, et al. (1995) Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein. Biophys J 69: 20–29.
[15]
Locke BC, MacInnis JM, Qian S, Gordon JI, Li E, et al. (1992) Fluorescence studies of rat cellular binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants. Biochemistry 31: 2376–2383.
[16]
Jin L, Abrahams JP, Skinner R, Petitou M, Pike RN, et al. (1997) The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci U S A 94: 14683–14688.
[17]
Langdown J, Johnson DJ, Baglin TP, Huntington JA (2004) Allosteric activation of antithrombin critically depends upon hinge region extension. J Biol Chem 279: 47288–47297.
[18]
Olson ST, Bjork I (1992) Regulation of thrombin by antithrombin and heparin cofactor II. In: Berliner LJ, editor. Thrombin: Structure and Function. New York: Plenum Press. pp. 159–217.
[19]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities f protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
Grigoryev SA, Woodcock CL (1998) Chromatin structure in granulocytes. A link between tight compaction and accumulation of a heterochromatin-associated protein (MENT). J Biol Chem 273: 3082–3089.
[22]
Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr 59: 1131–1137.
[23]
Potterton L, McNicholas S, Krissinel E, Gruber J, Cowtan K, et al. (2004) Developments in the CCP4 molecular-graphics project. Acta Crystallogr D Biol Crystallogr 60: 2288–2294.
