[Methods in Enzymology] Chromatin and Chromatin Remodeling Enzymes, Part B Volume 376 || Use of Nuclear Magnetic Resonance Spectroscopy to Study Structure-Function of Bromodomains

important issue that will require structural studies of relevant HAT com-plexes. These structures may be at the heart of understanding how HATactivity is coordinated with other histone-modifying activities to faithfullymodulate transcriptional regulation.

Acknowledgments

I would like to acknowledge all past and present members of my laboratory who have

contributed to the studies discussed in this chapter. In particular, I would like to thank

A. Clements, M. Holbert, A. Poux, J. Rojas, T. Sikorski, R. Trievel, and Y. Yan.

[8] use of nuclear magnetic resonance spectroscopy 119

[8] Use of Nuclear Magnetic Resonance Spectroscopyto Study Structure-Function of Bromodomains

By Shiraz Mujtaba and Ming-Ming Zhou

Characterization of the evolutionarily conserved protein modulardomains in signaling proteins that recognize post-translationally modifiedamino acids or unique sequence motifs in a protein has revolutionizedour understanding of regulation of protein-protein interactions or enzymeactivities in signal transduction that govern cell growth, proliferation,differentiation, and apoptosis.1 Chromatin remodeling represents anotherimportant frontier in cell biology.2,3 While recent studies have identifiednumerous conserved protein modules in many proteins and enzymeslinked to chromatin remodeling,3–5 their detailed molecular mechanismsremain elusive. Nuclear magnetic resonance (NMR) spectroscopy is apowerful tool not only for determination of high-resolution 3D structuresof protein domains but also for investigation of their biochemicalfunctions. The resulting structural and functional inferences can help usgain important insights into the molecular mechanisms underlying chroma-tin-mediated epigenetic control processes, including transcriptional acti-vation and repression, as well as gene silencing. Here we describe the

1 T. Pawson and P. Nash, Genes Dev. 14, 1027 (2000).2 R. D. Kornberg and Y. Lorch, Cell 98, 285 (1999).3 S. Bjorklund, G. Almouzni, I. Davidson, K. P. Nightingdale, and K. Weiss, Cell 96,

759 (1999).4 F. Jeanmougin, J. M. Wurtz, B. L. Douarin, P. Chambon, and R. Losson, Trends Biochem.

Sci. 22, 151 (1997).5 R. Aasland, T. J. Gibson, and A. F. Stewart, Trends Biochem. Sci. 20, 56 (1995).

Copyright 2004, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 376 0076-6879/04 $35.00

120 chromatin proteins [8]

procedures recently used to delineate structure-function relationships ofthe bromodomains.

The bromodomain of � 110 amino acids, first reported in the Drosophilaprotein brahma (hence the name),6,7 represents an extensive family of evo-lutionarily conserved protein modules found in many chromatin-associatedproteins and in nearly all known nuclear histone acetyltransferases(HATs).4 It has been long implicated from yeast genetic and biochemicalstudies that bromodomains play an important role in chromatin remodel-ing8–10 on the basis of their importance in the assembly and activity of mul-ti-protein complexes of chromatin remodeling,11,12 as well as by the factthat the bromodomain module is indispensable for the function of GCN5in yeast.13,14 For example, it has been shown that deletion of a bromodo-main in human HBRM, a protein in the SWI/SNF remodeling complex,causes both decreased stability and loss of nuclear localization.15,16 Bromo-domains of Bdf1, a Saccharomyces cerevisiae protein, are required for spor-ulation and normal mitotic growth.17 Finally, bromodomain deletion inSth1, Rsc1, and Rsc2, three members of the nucleosome remodelingcomplex, can cause a conditional lethal phenotype (in Sth1)18 or a strongphenotypic inhibition on cell growth (in Rsc1 and Rsc2).19 Notably, thephenotypic effect observed in Rsc1 and Rsc2 results from deletion ofonly the second but not the first bromodomain, suggesting that these twobromodomains serve distinct functions through interactions with different

6 J. W. Tamkun, R. Deuring, M. P. Scott, M. Kissinger, A. M. Pattatucci, T. C. Kaufman, and

J. A. Kennison, Cell 68, 561 (1992).7 S. R. Haynes, C. Dollard, F. Winston, S. Beck, J. Trowsdale, and I. B. Dawid, Nucleic Acids

Res. 20, 2603 (1992).8 J. E. Brownell, J. Zhou, T. Ranalli, R. Kobayashi, D. G. Edmondson, S. Y. Roth, and

C. D. Allis, Cell 84, 843 (1996).9 P. Filetici, C. Aranda, A. Gonzalez, and P. Ballario, Biochem. Biophys. Res. Commun. 242,

84 (1998).10 G. A. Marcus, N. Silverman, S. L. Berger, J. Horiuchi, and L. Guarente, EMBO J. 13,

4807 (1994).11 C. E. Brown, L. Howe, K. Sousa, S. C. Alley, M. J. Carozza, S. Tan, and J. L. Workman,

Science 292, 2333 (2001).12 D. E. Sterner, P. A. Grant, S. M. Roberts, L. J. Duggan, R. Belotserkovskaya, L. A. Pacella,

F. Winston, J. L. Workman, and S. L. Berger, Mol. Cell. Biol. 19, 86 (1999).13 T. Georgakopoulos, N. Gounalaki, and G. Thireos, Mol. Gen. Genet. 246, 723 (1995).14 P. Syntichaki, I. Topalidou, and G. Thireos, Nature 404, 414 (2000).15 C. Muchardt, B. Bourachot, J. C. Reyes, and M. Yaniv, EMBO J. 17, 223 (1998).16 C. Muchardt and M. Yaniv, Semin. Cell. Dev. Biol. 10, 189 (1999).17 P. Chua and G. S. Roeder, Mol. Cell. Biol. 15, 3685 (1995).18 J. Du, I. Nasir, B. K. Benton, M. P. Kladde, and B. C. Laurent, Genetics 150, 987 (1998).19 B. R. Cairns, A. Schlichter, H. Erdjument-Bromage, P. Tempst, R. D. Kornberg, and

F. Winston, Mol. Cell 4, 715 (1999).


biological ligands.19 The recent NMR-based structure-function analysis ofthe prototypical bromodomain from the transcriptional coactivator PCAF(p300/CBP-associated factor) demonstrates that bromodomains functionas acetyl lysine–binding domains,20 which offers insights into the molecularbasis of biological functions of bromodomains in a wide variety of cellularevents, including chromatin remodeling and transcriptional activation.21–24

In this chapter, we describe use of the NMR-based methods to studystructure and function of bromodomains, which is generalizable for otherconserved protein modular domains in chromatin remodeling.

Preparation of Protein Samples

Protein Expression and Stable Isotope Labeling

The cDNA construct that encodes the bromodomain of PCAF (residues719–832) used in the NMR structural analysis is designed on the basis ofsequence analysis, which shows that this region of PCAF contains the con-served bromodomain present in many other proteins.4,20 The expressionconstruct is ligated into the pET14b vector (Novagen) between Nde1 andBamH1 sires. The recombinant protein expressed in Escherichia coli BL21(DE3) cells contains a hexahistidine tag at its amino terminus followed by athrombin cleavage site. After confirming the clone by DNA sequencing,protein expression studies are conducted in E. coli BL21 (DE3) cells. Ini-tial optimization of experimental conditions for expression and solubility ofthe protein is conducted with small- and large-scale culture in Luria-Bertani (LB) media. Subsequently, protein samples for the NMR structuralstudy is expressed in an M9 minimal medium. Ingredients of the minimalmedium consist of NaCl (0.5 g/L), NH4Cl (1 g/L), KH2PO4 (3 g/L), andNa2HPO4-H2O (6 g/L), which after sterilization by autoclave are added toglucose (4 g/L), vitamin B1 (0.0005%), MgSO4 (1 �M), CaCl2 (100 �M)and ampicillin (100 mg/L). For a typical protein expression experiment,bacterial BL21(DE3) cells transformed with pET14b-bromodomain aregrown overnight at 30

�in 100 ml of the M9 minimal medium containing

ampicillin (100 mg/L). This starter culture is used to inoculate 1 L of freshM9 media, which is then incubated at 30

�for a few hours until OD600

reaches about 0.5. Protein expression is induced by adding 200–400 �M

20 C. Dhalluin, J. E. Carlson, L. Zeng, C. He, A. K. Aggarwal, and M.-M. Zhou, Nature 399,

491 (1999).21 M. H. Dyson, S. Rose, and L. C. Mahadevan, Front Biosci. 6, 853 (2001).22 F. Winston and C. D. Allis, Nat. Struct. Biol. 6, 601 (1999).23 B. D. Strahl and C. D. Allis, Nature 403, 41 (2000).24 L. Zeng and M.-M. Zhou, FEBS Lett. 513, 124 (2001).


isopropyl-�-d-thiogalactopyranoside (IPTG) to the cell culture, which isfurther incubated for another 10–12 h at 18

�. Cells are harvested by centri-

fugation at 3000g for 30 min at 4�, and cell pellets are collected for protein

purification (see later) or quick freezing in liquid nitrogen for storage at80

�. This expression procedure is also used to make various stable

isotope (15N, 13C/15N or 2H/13C/15N)-labeled proteins for the NMR struc-tural study. Particularly, the labeled proteins are prepared from bacterialcells grown in a minimal medium containing 15NH4Cl with or without13C6-glucose in H2O or 2H2O. 15NH4Cl and 13C6-glucose provide solenitrogen and carbon sources for recombinant protein expressed in E. coli.

Protein Purification

The harvested bacterial cells are resuspended in a lysis buffer [50 mMTris-HCl of pH 8.0, containing 10% glycerol, 1% NP-40, 300 mM NaCl,1 mM PMSF, and EDTA free protease inhibitors (one tablet per liter ofcell culture) (Roche)] and subjected to sonication. Cellular debris is re-moved by centrifugation at 100,000g for 20 min, and the supernatantobtained is used for subsequent protein purification.

The bromodomain protein is first purified by affinity chromatographyon a nickel-IDA column (Invitrogen) using an FPLC system (AmershamPharmacia Biosciences). The cell lysate is applied to the nickel resincolumn of 5 ml (for 3–4 L of cell culture) that is pre-equilibrated in a bind-ing buffer [50 mM Tris-HCl of pH 8.0, containing 250 mM NaCl, 5 mM�-mercaptoethanol (�-ME), 1 mM PMSF, and protease inhibitors], andsubsequently washed with 10–20 column volumes of the binding buffer,followed by 10 column volumes of a washing buffer [50 mM Tris-HCl ofpH 8.0, containing 250 mM NaCl, 5 mM �-ME, 1 mM PMSF, and proteaseinhibitors, plus 20 mM imidazole]. The hexahistidine-tagged bromodomainprotein is eluted from the column in 1.0-ml fractions with an elution bufferwith an 20–500-mM imidazole gradient in 50 mM Tris-HCl of pH 8.0, con-taining 250 mM NaCl, 5 mM �-ME, 1 mM PMSF, and protease inhibitors.Fractions containing the pure protein are pooled and dialyzed to a throm-bin cleavage buffer [50 mM Tris-HCl of pH 8.0, containing 250 mM NaCl,and 5 mM �-ME]. The hexahistidine tag in the recombinant protein is re-moved with thrombin treatment (�1 unit thrombin/mg protein) overnightat 4

�. The thrombin cleavage reaction is stopped by addition of 1 mM

PMSF. The protein sample is concentrated and applied to a size exclusionchromatography column for further purification using an FPLC system in aphosphate buffer [100 mM phosphate of pH 6.5, containing 0.5 mM EDTAand 1 mM DTT]. Peak fractions are collected, concentrated to �0.5 mM,and dialyzed to the final NMR buffer in H2O/2H2O (9:1) consisting of


100 mM phosphate, pH 6.5, containing 0.5 mM EDTA, 5 mM perdeuter-ated DTT. Typically, 5–10 mg of pure bromodomain protein is obtainedfrom 1 L of cell culture.

Protein Structure Determination by NMR

Three-dimensional structure of a protein can be determined by usingheteronuclear multi-dimensional NMR methods.25 The heteronuclearNMR methods separate the proton signals of a protein in the NMR spectraaccording to chemical shifts of their attached heteronuclei (such as 15N and13C), thus minimizing signal overlapping problems in the protein spectra.Also, NMR resonance assignments of the protein can be obtained in asequence-specific manner, which assures the accuracy of data analysisfor high-resolution structure determination. In addition, because of favor-able 1H, 13C, and 15N relaxation rates caused by partial deuteration ofthe protein, factional deuteration in combination with 13C and 15N-labelingis often employed for protein structure determination by NMR.26,27 Allthe NMR data are processed and analyzed using NMR software programsof NMRPipe28 and NMRView.29 For sequential backbone and side chainassignments and structure determination of the protein, a set of NMRexperiments is described briefly later.

Backbone Assignments. Sequence-specific backbone assignment isachieved using a suite of deuterium-decoupled 3D NMR experiments thatinclude HNCA, HN(CO)CA, HN(CA)CB, HN(COCA)CB, HNCO, andHN(CA)CO experiments.30 Using the triple-labeled (75% 2H, 13C, and15N) protein sample, we perform constant-time experiments to gain higherdigital resolution and use a water flip-back scheme to minimize amidesignal attenuation from water exchange.

Side-Chain Assignments. Sequential side-chain assignments areaccomplished from a series of 3D NMR experiments with alternativeapproaches to confirm the assignments. These experiments include 3D15N-edited TOCSY-HSQC, HCCH-TOCSY, (H)C(CO)NH-TOCSY, andH(C)(CO)NH-TOCSY.25

25 G. M. Clore and A. M. Gronenborn, Meth. Enzymol. 239, 249 (1994).26 M. Sattler and S. W. Fesik, Structure 4, 1245 (1996).27 M.-M. Zhou, K. S. Ravichandran, E. T. Olejniczak, A. P. Petros, R. P. Meadows, M. Sattler,

J. E. Harlan, W. Wade, S. J. Burakoff, and S. W. Fesik, Nature 378, 584 (1995).28 F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, and A. Bax, J. Biomol. NMR 6,

277 (1995).29 B. A. Johnson and R. A. Blevins, J. Biomol. NMR 4, 603 (1994).30 T. Yamazaki, W. Lee, C. H. Arrowsmith, D. R. Mahandiram, and L. E. Kay, J. Am. Chem.

Soc. 116, 11655 (1994).


NOE Analysis/Distance Restraints. Distance restraints are obtainedfrom analysis of 15N- and 13C-edited 3D NOESY data, which are collectedwith different mixing times to minimize spin diffusion problems. Thenuclear Overhauser effect (NOE)-derived restraints are categorized asstrong (1.8–3 A), medium (1.8–4 A), or weak (1.8–5 A) based on the ob-served NOE intensities. We also employ the recently developed ARIAprogram31 that is integrated with X-PLOR for the iterative automatedNOE analysis. ARIA-assigned NOE peaks are manually checked and con-firmed to ensure the success of ARIA/X-PLOR-assisted NOE analysis andstructure calculations.

Slow Exchange Amides. Amide protons involved in hydrogen bonds areidentified from an analysis of the amide exchange rates measured from aseries of 2D 1H/15N-heteronuclear single quantum coherence (HSQC)spectra recorded after adding 2H2O to the protein sample.

Stereospecific Methyl Groups. Stereospecific assignments of methylgroups of Val and Leu residues are obtained from an analysis of carbonsignal multiplet splitting using 10% 13C-labeled protein sample, whichcan be readily prepared using 10% 13C-glucose containing M9 minimalmedium.32

Dihedral Angle Restraints. � angle constraints are generated from the3JHNH� coupling constants measured in a 3D HNHA-J experiment.33

Stereospecific assignments of �-methylene protons, which give informationon �1, angles can be obtained from HNHB34 and 15N-edited TOCSY witha short mixing time.35

Structure Calculations and Refinements. Structures of the protein arecalculated using a distance geometry/simulated annealing protocol withthe X-PLOR program.36–38 The structure calculations employ inter-protondistance restraints obtained from 15N- and 13C-resolved NOESY spectra ofthe protein or protein/peptide complex. The initial structures are typicallycalculated with only manually assigned NOE-derived distance restrains,which are used to assist further NOE assignments and identify hydrogenbond partners for the slow exchange amide protons. The converged struc-tures are refined with the experimental restraints of dihedral angles and

31 M. Nilges and S. O’Donoghue, Prog. NMR Spectrosc. 32, 107 (1998).32 D. Neri, T. Szyperski, G. Otting, H. Senn, and K. Wuthrich, Biochemistry 28, 7510 (1989).33 G. Vuister and A. Bax, J. Am. Chem. Soc. 115, 7772 (1993).34 J. C. Matson, O. W. Sorensen, P. Soresen, and F. M. Poulsen, J. Biomol. NMR 3, 239 (1993).35 G. M. Clore, A. Bax, and A. M. Gronenborn, J. Biomol. NMR 1, 13 (1991).36 A. T. Brunger, ‘‘X-PLOR Version 3.1: A System for X-Ray Crystallography and NMR.’’

Yale University Press, New Haven, CT, 1993.37 J. Kuszewski, M. Nilges, and A. T. Brunger, J. Biolmol. NMR 2, 33 (1992).38 M. Nilges, G. M. Clore, and A. M. Gronenborn, FEBS Lett. 229, 317 (1988).


hydrogen bonds. Final structure calculations employ the manual and theARIA-assisted NOE distance restraints, together with hydrogen bond dis-tance restraints and dihedral angle restraints. The distance restraint forceconstant used in the calculations is typically 50 kcal/mol/A 2, and no NOEis violated by more than 0.3 A. The torsion restraint force constant is200 kcal/mol/rad2, and no dihedral angle restraint is violated by more than5�. Only the covalent geometry terms, NOE, torsion, and repulsive van der

Waals terms are used in the structure refinement. A large, and negativeLennard-Jones potential energy should be observed for the final structures,indicating good non-bonded geometry of the structure. Procheck39 analysisis also performed to show the majority of the protein residues that are inpreferred and allowed regions of the Ramachandran map. Finally, struc-tures of proteins determined using NOE-derived distance restraints anddihedral angle restraints can be further refined with use of residual dipolarcouplings, which can be measured in bicelle-based liquid crystalline orcross-linked polyacrylamide gel medium and implemented in the finalrefinement stage of the structure calculations.40–43

The Bromodomain Structure

The 3D structure of a prototypical bromodomain from the transcrip-tional coactivator PCAF determined by NMR shows that the bromodo-main adopts an atypical left-handed four-helix bundle (helices �Z, �A,�B, and �C) (Fig. 1A).20 A long intervening loop between helices �Z and�A (termed the ZA loop) is packed against the loop connecting helices Band C (named the BC loop) to form a surface accessible hydrophobicpocket, which is located at one end of the four-helix bundle, opposite theamino and carboxy termini of the protein. Mutagenesis studies suggest thattertiary contacts among the hydrophobic and aromatic residues betweenthe two inter-helical loops contribute directly to the structural stability ofthe protein.20 This unique structural fold is highly conserved in the bromo-domain family, as supported by several more recently determined struc-tures of bromodomains from human GCN544 and S. cerevisiae GCN5p45

39 R. A. Laskowski, J. A. Rullmannn, M. W. MacArthur, R. Kaptein, and J. M. Thornton,

J. Biomol. NMR 8, 477 (1996).40 J. H. Prestegard, Nat. Struct. Biol. 5 Suppl., 517 (1998).41 J. J. Chou, S. Li, C. B. Klee, and A. Bax, Nat. Struct. Biol. 8, 990 (2001).42 S. Cavagnero, H. J. Dyson, and P. E. Wright, J. Biomol. NMR 13, 387 (1999).43 J. J. Chou, S. Gaemers, B. Howder, J. M. Louis, and A. Bax, J. Biomol. NMR 21, 377 (2001).44 B. P. Hudson, M. A. Martinez-Yamout, H. J. Dyson, and P. E. Wright, J. Mol. Biol. 304,

355 (2000).45 D. J. Owen, P. Ornaghi, J. C. Yang, N. Lowe, P. R. Evans, P. Ballario, D. Neuhaus,

P. Eiletici, and A. A. Travers, EMBO J. 19, 6141 (2000).

Fig. 1. Ligand binding of PCAF bromodomain. (A) Three-dimensional NMR structure

of the PCAF bromodomain. (B) Superimposed region of the 2D 15N-HSQC spectra of the

bromodomain (� 0.5 mM) in its free form (dark) and complexed to a histone H4 peptide

containing acetylated lysine 8 (SGRGKGG-AcK-GLGK, where AcK is acetyl-lysine) (molar

ratio 1:6) (light). The movement of protein resonances upon ligand binding is indicated by

arrows connecting from the free to the ligand-bound forms. (C) Ribbon and dotted-surface

diagram of the bromodomain depicting the location of the lysine-acetylated H4 peptide-

binding site. Bromodomain residues that exhibit major chemical shift changes of the backbone

amide 1H and 15N resonances upon binding to the AcK histone H4 peptide as observed in the

2D 15N-HSQC spectra are indicated in lighter color. Note that most of these perturbed

residues are located in the ZA and BC loops. [From C. Dhalluin, J. E. Carlson, L. Zeng, C. He,

A. K. Aggarwal, and M.-M. Zhou, Nature 399, 491 (1999).]



as well as the double bromodomain module of human TAFII250.46 Thestructural similarity among these bromodomains is very high for the fourhelices with pairwise root-mean-square deviations of 0.7–1.8 A for thebackbone C� atoms. The majority of structural deviations are localized inthe loop regions, particularly in the ZA and BC loops. This observationis in an agreement with relatively high sequence variations in these loops.4

The modular structure supports the notion that bromodomains act as afunctional unit for protein interactions, and multiple bromodomainmodules can be placed sequentially in a protein to serve similar or distinctfunctions.4,20

Ligand Binding Study by NMR

The unique advantage of protein structural analysis by NMR is that inaddition to determination of a 3D structure of a protein, resonance assign-ments obtained in the structural study provide a map of the entire proteinat atomic details-level, which could be used in biochemical analysis of pro-tein-ligand interactions. Because the NMR resonances of protein residuesare highly sensitive to local chemical and conformational changes, bindingof a ligand to a host protein could be detected by resonance perturbationsof protein residues directly or indirectly involved in interactions with theligand.47,48 For highly specific interactions between a host protein and aligand, protein resonances change as a function of ligand concentrationuntil complete saturation of the protein by the ligand. NMR titration ofprotein and ligand binding, therefore, can be used to determine binding af-finity (dissociation constant, KD) of the complex. The ligand-binding studyis most conveniently performed by using 2D 15N-HSQC spectra that recordbackbone amide proton and nitrogen resonances of a uniformly 15N-labeled protein. Because the ligand is typically not 15N-labeled thus invis-ible in 15N-HSQC spectra, protein resonance changes in the spectra arerelatively easy to monitor. With the resonance assignment of a protein,using this NMR method, one can identify the location of the ligand-bindingsites on the protein. The NMR titration study of the PCAF bromodomainshows that the protein can bind to acetyl-lysine–containing peptidesderived from major acetylation sites on histones H3 and H4 in a highlyspecific manner, and the interaction is dependent on acetylation oflysine (Fig. 1B and C).20

46 R. H. Jacobson, A. G. Ladurner, D. S. King, and R. Tjian, Science 288, 1422 (2000).47 P. J. Hajduk, R. P. Measdows, and S. W. Fesik, Q. Rev. Biophys. 32, 211 (1999).48 J. M. Moore, Curr. Opin. Biotech. 10, 54 (1999).


This unique ability of NMR spectroscopy to detect relatively weak buthighly specific interactions between a host protein and a ligand is also dem-onstrated by our recent study of the bromodomain of transcriptional coac-tivator CBP (CREB-binding protein) and the tumor suppressor proteinp53. It is known that transcriptional activity of p53 in cell cycle arrest, se-nescence or apoptosis is tightly controlled by acetylation of its C-terminallysines, that is, K320 by PCAF,49 and K373 and K382 by CBP,50 whichresults in its association with transcriptional coactivators includingCBP.51,52 Our study shows that p53/CBP association involves CBP bromo-domain binding to p53 at the acetylated lysine 382 but not K373 or K320,which can be demonstrated by NMR titration with lysine-acetylated p53peptides containing different acetylation sites (Mujtaba and Zhou, unpub-lished results). Notably, amino acid sequences of the acetylated K373 andK382 peptides used in the NMR-binding study are identical except that theacetylated lysine is in a different position. Remarkably, only the latter p53peptide causes ligand concentration–dependent resonance perturbations ofprotein residues in 2D 15N-HSQC spectra of the bromodomain, underscor-ing the highly selective nature of the CBP bromodomain/p53 AcK382 rec-ognition. None of the p53 peptides showed any detectable binding to thestructurally homologous bromodomain from PCAF.20,53

To understand the detailed structural basis of molecular recognition ofacetyl-lysine–containing peptide, one can determine the 3D structure of abromodomain in complex with a synthetic, lysine-acetylated peptide de-rived from the known binding site in its biological binding partner protein.Intermolecular NOE distance restraints required for structure determin-ation of the complex can be obtained from a 13C-edited (F1) and 15N, and13C-filtered (F3) 3D NOESY spectrum collected for a sample containingisotope-labeled protein and unlabeled ligand.54 In addition, a 2D 13C/15N-filtered 1H-1H NOESY spectrum can provide intra-molecular NOEs forthe peptide molecule bound to the protein. Structure calculations of thecomplex using both inter- and intra-molecular NOE distance restraints to-gether with other experimentally determined hydrogen bond distancerestraints and dihedral angle restrains are similar to those described earlier

49 L. Liu, D. M. Scolnick, R. C. Trievel, H. B. Zhang, R. Marmorstein, T. D. Halazonetis, and

S. L. Berger, Mol. Cell Biol. 19, 1202 (1999).50 W. Gu and R. G. Roeder, Cell 90, 595 (1997).51 N. A. Barlev, L. Liu, N. H. Chehab, K. Mansfield, K. G. Harris, T. D. Halazonetis, and S. L.

Berger, Mol. Cell 8, 1243 (2001).52 C. Prives and J. L. Manley, Cell 107, 815 (2001).53 S. Mujtaba, Y. He, L. Zeng, A. Farooq, J. E. Carlson, M. Ott, E. Verdin, and M.-M. Zhou,

Mol. Cell 9, 575 (2002).54 M. Sattler, J. Schleucher, and C. Griesinger, Prog. NMR Spectrosc. 34, 93 (1999).


for the structure determination of the protein alone using XPLORprogram.

Protein–Peptide Binding Assays

Recent structural studies of different bromodomains show that bromo-domains share a conserved left-handed four-helix bundle fold, and acetyl-lysine–containing peptides are bound between the ZA and BC loops.Residues important for acetyl-lysine recognition in different bromodo-mains are conserved; however, ligand selectivity differ due to a few but im-portant differences in bromodomain sequences. These include variations inthe ZA loops, which have relatively low sequence conservation and aminoacid deletion or insertion in different bromodomains; and differences inbromodomain residues that directly interact with residues surroundingacetyl-lysine in a target protein. Structure-based mutational analysis canbe employed to determine the structural basis of molecular recognition ofa bromodomain/ligand complex. For example, in an effort to determine themolecular determinants of the selective recognition between the PCAFbromodomain and HIV-1 Tat at the acetylated lysine 50, we performedstructure-based mutational analysis of the complex in an in vitro–bindingassay using the recombinant and purified GST-fusion bromodomain andan N-terminal biotinylated, p53 peptide containing lysine-acetylated K382that is immobilized onto Streptavidin agarose beads.53

The cDNA encoding GST-fusion bromodomain of PCAF is cloned intothe pGEX4T-3 vector (Amersham Biosciences). The GST bromodomain isexpressed in E. coli BL21(DE3) codon-plus cells using a procedure similarto that described earlier for NMR protein sample preparation, except thatthe GST protein is prepared in LB medium. The harvested bacterial cellsculture is re-suspended in lysis buffer [20 mM Tris of pH 8.0, 150 mMNaCl, 1.0% NP-40, 1 mM PMSF, 5 mM �-ME, 10% glycerol, 5 mMEDTA, 1 mM PMSF, and DNAse]. After cell lysis by sonication and re-moval of cell debris by centrifugation, the supernatant is purified by affinitychromatography using a glutathione Sepharose resins according to manu-facturer’s (Amersham Bioscience) instruction. The binding assay is per-formed by incubating an equal amount of the PCAF bromodomain withbiotinylated HIV Tat peptide for 2 h at 22

�in the binding buffer (50 mM

Tris buffer of pH 7.5, containing 50 mM NaCl, 0.1% BSA, and 1 mMDTT). The protein-peptide complex is pulled down by 10 �l of Streptavi-din agarose (Novagen) by further incubation of 30 min. To minimizenon-specific binding, the beads are washed extensively (thrice) with a highsalt- and detergent-containing washing buffer (50 mM Tris buffer of pH7.5, containing 300 mM NaCl, 0.1% NP-40, and 1 mM DTT). Proteins


eluted from the agarose beads are separated by SDS-PAGE and protein/peptide interaction is visualized by western blotting using anti-GST anti-body (Sigma) and horseradish-peroxidase–conjugated goat anti-rabbit IgG.

Such a GST pull-down assay is also used to assess other bromodomains’binding or effect of site-directed mutation of PCAF bromodomain residueson protein binding to the HIV-1 Tat peptide. Furthermore, this bindingassay can be used for mutational analysis of Tat peptide residues in apeptide competition assay, in which a non-biotinylated peptide carrying amutation at a specific amino acid residue competes with the biotinylatedwild-type Tat AcK50 peptide for binding to the GST-fusion PCAF bromo-domain. The molar ratio of the mutant and wild-type peptides in the mix-ture is kept at 1:2. Because of the high sensitivity of western blottingdetection, this binding study can be performed at a protein concentration(10 �M) much lower than that required for NMR study (�200 �M), thusensuring specificity of protein-peptide interactions. Using this GST pull-down–based assay, mutational analyses of the protein and peptide residuesvalidate the molecular interactions observed in the NMR structure ofthe complex, and identify key residues of the PCAF bromodomain thatare important for recognition of the acetyl-lysine and its flanking residuesin the HIV-1 Tat peptide.

Perspective

The NMR-based methods described here have enabled establishmentof the biochemical functions of bromodomains as acetyl-lysine–bindingdomains.20,44,53 This new mechanism of regulating protein-protein inter-actions via lysine acetylation has broad implications in a wide varietyof cellular processes, including chromatin remodeling and transcriptionalactivation.21–23,53,55 Such a powerful NMR structure-based approach isreadily applicable to investigate the biological functions of the other evolu-tionarily conserved protein modular domains that play an important role inregulation of chromatin remodeling.

Acknowledgment

The work was supported by a grant from the National Institutes of Health to M.-M. Z.

(CA87658).

55 A. Dorr, V. Kiermer, A. Pedal, H.-R. Rackwitz, P. Henklein, U. Schubert, M.-M. Zhou,

E. Verdin, and M. Ott, EMBO J. 21, 2715 (2002).

Documents

[Methods in Enzymology] Chromatin and Chromatin Remodeling Enzymes, Part B Volume 376 || Use of Nuclear Magnetic Resonance Spectroscopy to Study Structure-Function of Bromodomains