Disruption of Bcr-Abl Coiled Coil Oligomerization by Design*□S

Received for publication, May 25, 2011 Published, JBC Papers in Press, June 9, 2011, DOI 10.1074/jbc.M111.264903

Andrew S. Dixon‡, Scott S. Pendley‡, Benjamin J. Bruno‡, David W. Woessner§, Adrian A. Shimpi¶,Thomas E. Cheatham III‡�, and Carol S. Lim‡1

From the Departments of ‡Pharmaceutics and Pharmaceutical Chemistry, §Pharmacology and Toxicology, and �MedicinalChemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108 and ¶Juan Diego Catholic High School,Draper, Utah 84020

Oligomerization is an important regulatory mechanism formany proteins, including oncoproteins and other pathogenicproteins. The oncoprotein Bcr-Abl relies on oligomerization viaits coiled coil domain for its kinase activity, suggesting that adesigned coiled coil domain with enhanced binding to Bcr-Abland reduced self-oligomerization would be therapeutically use-ful. Key mutations in the coiled coil domain of Bcr-Abl wereidentified that reduce homo-oligomerization through intermo-lecular charge-charge repulsion yet increase interaction withthe Bcr-Abl coiled coil through additional salt bridges, resultingin an enhanced ability to disrupt the oligomeric state of Bcr-Abl.The mutations were modeled computationally to optimize thedesign. Assays performed in vitro confirmed the validity andfunctionality of the optimal mutations, which were found toexhibit reduced homo-oligomerization and increased bindingto the Bcr-Abl coiled coil domain. Introduction of the mutantcoiled coil into K562 cells resulted in decreased phosphoryla-tion of Bcr-Abl, reduced cell proliferation, and increasedcaspase-3/7 activity and DNA segmentation. Importantly, themutant coiled coil domain was more efficacious than the wildtype in all experiments performed. The improved inhibition ofBcr-Abl througholigomeric disruption resulting from thismod-ified coiled coil domain represents a viable alternative to smallmolecule inhibitors for therapeutic intervention.

Coiled coil domains are ubiquitous protein structural motifsfound in�10% of all eukaryotic proteins (1). Characterized by aheptad repeat (sequence of seven amino acids) and associationof two or more �-helices, coiled coils provide oligomerizationcapabilities (both homo- and hetero-oligomerization) usefulfor structural scaffolding and protein recognition. Coiled coildomains are critical for regulating many processes involvedin the pathogenesis of various diseases (2). Thus, rationally

designed coiled coils can be used as therapeutics by interferingwith the activity of a pathogenic protein through oligomericdisruption. One example is enfuvirtide (Fuzeon�), a fusioninhibitor that disrupts the helix bundle formation necessary forHIV-1 viral entry (3), spawning the next generation of rationallydesigned fusion inhibitors (4–8). The coiled coil reported herewas designed to bind to the target (Bcr-Abl) better than thetarget protein binds to itself while exhibiting minimalhomo-oligomerization.Coiled coils are very well characterized, and the high corre-

lation between their sequence and structure (9) is advantageousfor rational design. Coiled coil oligomerization involves hydro-phobic interactions, salt bridge formation, and helicity. In theheptad repeat, hydrophobic packing at the “a” and “d” positions(see Fig. 1, green residues) helps drive association with furtherstability provided by the residues at positions “g” and “e,” whichare commonly charged and interact to form salt bridges (seeFig. 1, red and blue residues). Because charged residues are rou-tinely found at the g and e positions, mutating these residues ina rational manner to add salt bridges to favor formation ofhetero-oligomers (10–12) and charge-charge repulsions toreduce the formation of homo-oligomers (13, 14) can changethe affinity and specificity of the coiled coil dimer.Bcr-Abl exists primarily as a tetramer (more specifically as a

dimer of dimers), facilitating the trans-autophosphorylationnecessary to activate the tyrosine kinase domain (15, 16). Olig-omerization of Bcr-Abl is achieved through a coiled coildomain at the N terminus of the protein. Bcr-Abl constructslacking the N-terminal coiled coil fail to induce chronicmyelogenous leukemia (CML)3 in amurinemodel (15, 17), thussetting the stage for oligomeric disruption as a therapy. Proof ofconcept for using the coiled coil domain to inhibit Bcr-Ablactivity has already been demonstrated through retroviraltransfection of the wild-type coiled coil domain or addition ofthe purified protein attached to a cytoplasmic transductionpeptide (18–22). To further this approach, we designed andtested amutant coiled coil (CCmut2) using both computationaland in vitro experiments. ThisCCmut2 disfavors self-oligomer-ization (CCmut2:CCmut2) yet appears to bind more tightly to

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA129528 (to C. S. L.) and GM079383 (to T. E. C.). This work was alsosupported by an American Foundation for Pharmaceutical Education pre-doctoral fellowship (to A. S. D), Graduate Research Fellowship, Universityof Utah (to A. S. D.) the ALSAM Foundation (to A. A. S), and National ScienceFoundation Grant TG-MCA01S027 (to T. E. C.; for computer time).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 To whom computational correspondence should be addressed. E-mail:tec3@utah.edu.

2 To whom all other correspondence should be addressed: Dept. of Pharma-ceutics and Pharmaceutical Chemistry, College of Pharmacy, University ofUtah, 421 Wakara Way, Rm. 318, Salt Lake City, UT 84108. Fax: 801-585-3614; E-mail: carol.lim@pharm.utah.edu.

3 The abbreviations used are: CML, chronic myelogenous leukemia; CC, wild-type coiled coil domain from Bcr-Abl; CCmut1, mutant coiled coil domainwith S41R, L45D, E48R, V49D, and Q60E mutations; CCmut2, mutant coiledcoil domain with C38A, S41R, L45D, E48R, and Q60E mutations; AMBER,assisted model building with energy refinement; MD, molecular dynamics;MM-PBSA, molecular mechanics Poisson-Boltzmann/surface area; NTA,nuclear translocation assay; CFA, colony forming assay; PS, protein switch;CrkL, Crk-like; EGFP, enhanced GFP; ANOVA, analysis of variance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 31, pp. 27751–27760, August 5, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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the target Bcr-Abl coiled coil domain (CCmut2:Bcr-Abl) thanthe native oligomerization partner of the target (Bcr-Abl:Bcr-Abl) and resulted in superior inhibition of Bcr-Abl.The coiled coil domain from Bcr-Abl consists of 72 amino

acids composed of two �-helices (�1 (residues 5–15) and �2(residues 28–67)) that form an N-shaped configuration withtwo parallel helices connected by a short linker (see Fig. 1A)(23). Upon dimerization, the resulting coiled coil has an antipa-rallel orientation with �2 at the core and �1 latching onto thebackside of the opposing �2 helix (domain swapping) (1, 23).The majority of the dimer interface is composed of the classicknobs-in-holes type hydrophobic interactions from residues atthe a anddpositions (24). Further stabilization comes from fourinterchain salt bridges between residues in the �2 helices (Fig.1A) as well as packing of aromatic residues from the �1 helixand opposing �2 helix. As seen in Fig. 1A, there are twouncharged residues (Ser-41 and Gln-60) that are in the appro-priate position for the formation of salt bridges with twocharged residues (Glu-32 and Glu-48). Thus, mutation ofSer-41 and Gln-60 to positively charged amino acids has thepotential to provide two additional salt bridges with Bcr-Abl(Fig. 1B) and thus enhance binding. However, although thisprovides more salt bridges in the hetero-oligomer, these muta-tions alone are undesirable as they allow the formation of agreater number of salt bridges in the homo-oligomer. Toreduce homo-oligomerization in the mutant coiled coil, resi-dues proximal to charged residues on the opposing helix wereconsidered as candidates for mutation to introduce charge-charge repulsion (13, 14). Leu-45 and Glu-48 were identified astwo such residues (Fig. 1C). In addition, previous reports haveincorporated a C38A mutation primarily for crystallizationpurposes (1, 23), and this mutation was also studied. Putativemutations were investigated first through molecular modelingand state-of-the art biomolecular simulation and free energyanalysis to ascertain the impact on the coiled coil structure andstability. Such in silico methods provide a means to efficiently(and inexpensively) assess the influence of mutation. Theresulting optimized mutant coiled coil (CCmut2) contains fivemutations (C38A, S41R, L45D, E48R, and Q60E) and was fur-ther assessed in vitro. Taken together, these results demon-strate the effectiveness of the mutant coiled coil domain and,importantly, further illustrate the ability to rationallymodify anexisting coiled coil domain to improve therapeutic efficacy.

EXPERIMENTAL PROCEDURES

ComputationalModeling and Simulation—The initialmodelstructure was obtained from the crystal structure (refined to2.2-Å resolution) of the N-terminal oligomerization domain ofBcr-Abl (Protein Data Bank code 1K1F, chains A and B) (23).Selenomethionine residues were mutated to methionine, andposition 39 was mutated back to cysteine to maintain consis-tency with the natural protein. Amino acid side chain muta-tions were introduced using DeepView (Swiss-PdbViewer) (26)and by hand with the LEAP module from AMBER 9 (27).Molecular dynamics (MD) and free energy simulations wereperformed to assess the structure and stability of the modelcoiled coil structures. Such methods have proven useful forreproducing and predicting the structure of proteins, including

the folding of small proteins and influence of amino acid sidechain substitutions (28–34). All simulations were completedusing the AMBER modeling suite (27) and the AMBER ff03protein force field (35) with explicit solvent and counter-ionsusing standard simulation protocols, including minimizationand �40–90 ns of MD sampling with Ewald treatments of theelectrostatics (36). For further detail, refer to the supplementaldata. Representative plots of the root-mean-squared deviationsto the initial structure of the coiled coil during the MD simula-tions can be found in supplemental Fig. S1. The MD trajectoryresults (effectively the time history of the atomicmotions of themodel structures at different intervals over the simulation)were analyzed with various tools. Quantification and compari-son of the relative helical content weremeasured by calculatingmean residue ellipticities at 222 nm (representative of helixcontent in circular dichroism (CD) spectra) of five individual500-ps average structures spanning the final 5 ns of simulationof each coiled coil dimer using the DichroCalc program (37).Structural helical content (or percent helicity defined as thenumber of residues in an �-helix divided by the total number ofresidues) was also calculated based on secondary structure asdetermined by peptide backbone � and � torsions from thefinal 10 ns of simulation using the DSSP method (38) as imple-mented in UCSF Chimera (39). Intrahelical hydrogen bonds(i.e. carbonyl oxygen of residue “i” to the amine nitrogen ofresidue “i � 4”) were calculated over the final 10 ns of MDwithdistances less than 3.5 Å indicative of a hydrogen bond. Toestimate the relative binding free energies of the coils in thedimers, MM-PBSA as implemented in AMBER (40) anddescribed by Gohlke and Case (41) was applied to independentMD trajectories for the dimer and individual monomers. Inaddition to the postprocessing of MD results, calculations ofthe relative free energy of binding with respect to the wild-typedimer (��Gbinding) were completed using more detailed ther-modynamic integration methods for the CCmut2 dimers (42).On the basis of a thermodynamic cycle (see supplemental Fig.S2), the relative free energy of binding can be calculated by“mutating” the original protein (� � 0) to incorporate designedamino acid side chain pointmutations (� � 1) in both the dimerand monomeric states over different � states in silico. Incorpo-ration of the five amino acid mutations considered using thisapproach was accomplished stepwise (see Fig. 1B). Two stepswere required to incorporate all five mutations to form the het-erodimer mutant (CC-CCmut2), and an additional two stepswere required to perturb the transition dimer into thehomodimer (CCmut2-CCmut2). Similarly, two stepswere usedto incorporate the five mutations in the unbound monomer.Relatively long (6-ns equilibration, �6-ns accumulation) MDsimulations were performed at each � for the thermodynamicintegration. Further technical details are provided in the sup-plemental data.Construction of Plasmids and Mutagenesis—The gene

encoding the coiled coil domain from Bcr-Abl (43) was ampli-fied via PCR with the primers 5�-tgtaactcgagttatggtggacccggt-g-3� and 5�-atgctctcgagaccggtcatagctcttc-3� and subcloned intothe XhoI site of the plasmid pEGFP-PS, an optimized proteinswitch (PS) (44, 45) for use in the nuclear translocation assay(46), and pEGFP-C1 (Clontech) for other experiments. These

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plasmids, named pPS-CC and pEGFP-CC, respectively, werethen used as the template DNA for site-directed mutagenesisusing the QuikChange II site-directed mutagenesis kit (Strat-agene, La Jolla, CA) to generate five amino acid mutations(S41R, L45D, E48R, V49D, andQ60E). The primers used for themutagenesis were 5�-ggagcgctgcaaggcccgcattcggcgcgacgag-cagcgggacaaccaggagcgcttccgcatgatctacctggagacgttgctggcca-agg-3� and the reverse complement. Another site-directedmutagenesis was carried out to make C38A and D49V muta-tions with the primers 5�-caggagctggagcgcgccaaggcccgcat-tcg-3� (and reverse complement) and 5�-gcgacgagcagcgggtgaa-ccaggagcgcttcc-3� (and reverse complement), respectively. Thefinal constructs were termed pEGFP-CCmut2 and pPS-CC-mut2. The primers 5�-cgcaagggagctcccatcatcatcatcatcatcttgaa-gttctttttcaaggtcctatggtggacccggtgggcttc-3� and 5�-agcatggatcc-ctaccggtcatagctcttcttttccttggc-3� were used to PCR amplifywild-type and mutant coiled coil domains, which were sub-cloned into pMAL-c2x (New England Biolabs, Ipswich, MA) atthe SacI and BamHI sites to generate pMAL-H6-PP-CC andpMAL-H6-PP-CCmut2 used for protein expression. The prim-ers 5�-tgtaactcgagttatggtggacccggtg-3� and 5�-atgctctcgagccgg-tcatagctcttc-3�were used to PCR amplify both coiled coil genes(wild type and mutant), and each was subsequently subclonedinto the XhoI site of pDsRed2-N1 (Clontech) to makepDsRed-CC and pDsRed-CCmut2. Similarly, the primers 5�-tgtaaggcccagccggccatggtggacccggtg-3� and 5�-cggggcgcggccg-cccggtcatagctcttcttttc-3� were used to insert the genes into theplasmid pEFVP16 (mammalian two-hybrid prey vector con-

taining the VP16 activation domain; obtained from Dr. T. H.Rabbitts, Leeds Institute of Molecular Medicine, Leeds, UK) atthe SfiI and NotI sites to generate pEFVP16-CC and pEFVP16-CCmut2. The primers 5�-tgtaagaattcatggtggacccggtg-3� and5�-atgctgaattcaccggtcatagctcttc-3� were used to insert thecoiled coils into the vector pM1 (mammalian two-hybrid baitvector containing the Gal4 binding domain; obtained from Dr.T. H. Rabbitts) at the EcoRI site to generate pM1-CC andpM1-CCmut2.Cell Lines and Transient Transfection—Cells were main-

tained in a 5% CO2 incubator at 37 °C. K562 (a kind gift fromKojo Elenitoba Johnson, University of Michigan) and COS-7cells (ATCC) were grown in RPMI 1640 medium (Invitrogen)supplemented with 10% FBS (HyClone Laboratories, Logan,UT), 1% penicillin-streptomycin (Invitrogen), 0.1% gentamicin(HyClone), and 1% L-glutamine (HyClone). K562 cells werepassaged every 2 days and maintained between 0.1 and 1 106cells/ml. Amaxa Nucleofector II (Lonza Group Ltd., Basel,Switzerland) was used to transfect 2 106 cells with 5–8 �g ofDNA in solutionV following themanufacturer’s recommendedprotocol and nucleofection program (T-013). COS-7 cells werepassaged every 2–3 days and transfected 24 h after seeding thecells using Lipofectamine LTX (Invitrogen) as recommendedby the supplier.Protein Purification and CD—Fusion proteins consisting of

maltose-binding protein (MBP), a His tag, a PreScission prote-ase site, and CC or CCmut2 were expressed in BL21star DE3Escherichia coli cells (Invitrogen) from pMAL-H6-PP-CC or

FIGURE 1. Ribbon diagrams (with corresponding helical wheel diagram below) of wild-type homodimer (A), wild-type-CCmut2 heterodimer (B), andCCmut2-CCmut2 homodimer (C). Gray ribbons (ribbon diagrams) or dots (helical wheel diagrams) represent the wild-type coiled coil domain, and cyanrepresents CCmut2. The side chains of key residues (Glu-34, Lys-39, Ser/Arg-41, Leu/Asp-45, Glu-46, Glu/Arg-48, Arg-53, Arg-55, and Gln/Glu-60) are shown asred (acidic), blue (basic), green (hydrophobic), yellow (serine), or black (glutamine) spheres (ribbon diagrams) or font (helical wheel diagrams). Dotted linesindicate possible ionic interactions, and solid lines indicate charge-charge repulsions. Ribbon diagrams were generated with UCSF Chimera starting with theBcr coiled coil domain crystal structure (Protein Data Bank code 1K1F).

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pMAL-H6-PP-CCmut2 plasmids. The proteins were purifiedover amylose resin (NewEngland Biolabs) with gravity flow andcleaved with PreScission protease (a kind gift from Dr. ChrisHill, University of Utah), and MBP-H6 was removed by purifi-cation over HisPur Cobalt resin (Thermo Scientific, Waltham,MA). Reverse phaseHPLCwas performed as a final purificationbefore lyophilizing the protein. CC and CCmut2 purified pro-teins were confirmed through mass spectroscopy (Mass Spec-troscopy and Proteomics Core Facilities, University of Utah).CD experiments were performed on anAviv 410 CD spectrom-eter (Aviv Biomedical Inc., Lakewood,NJ).Measurements from190 to 300 nm (1-nm steps) were taken on 10 �M protein solu-tions in PBS (50 mM sodium phosphate, 150 mM NaCl, 0.5 mM

DTT, pH 7.2) in a 1-mm-path length cuvette. 3-s equilibrationtimes were allowed prior to each measurement, and the signalwas averaged over 3 s. The average of three scans was used foreach solution. Thermal denaturation was monitored in a 1-cmcuvette at 222 nm every 2 °C from 10 to 95 °C and back down to10 °C in 10 °C steps. The protein concentrations used were 10�M CC, 10 �M CCmut2, and 5 �M CC � 5 �M CCmut2. Inaddition, a mixing cell cuvette was used with 2.5 �M CC in onechamber and 7.5�MCCmut2 in the other chamberwith spectraacquired prior to and aftermixing. Aftermixing and before dataacquisition, the sample was incubated at 80 °C for 10 min andthen allowed to re-equilibrate at 10 °C.Nuclear Translocation Assay—The nuclear translocation

assay (NTA) was performed as described previously (46).Briefly, this assay uses a nuclear localization-inducible proteinswitch fused to a protein of interest and measures its ability totranslocate a second protein into the nucleus. Here, the proteinfused to the protein switch was either the coiled coil domain ormutant coiled coil domain, and its ability to translocate acotransfected coiled coil domain or mutant coiled coil domaininto the nucleus wasmeasured. 24 h after transient transfectionof COS-7 cells, 200 nM dexamethasone or an equal volume ofethanol (carrier) was added to the cells and incubated for 1 h.0.5 �l of H33342 (nuclear dye) was added and incubated foranother 15 min before imaging the cells with a fluorescencemicroscope as described previously (43). The percentage ofprotein localized in the nucleus was quantified with and with-out ligand to determine the nuclear increase after ligandinduction.Mammalian Two-hybrid Assay—The pM1-CC or pM1-CC-

mut2 (bait), pEFVP16-CC or pEFVP16-CCmut2 (prey), pG5-Fluc (reporter; obtained from Dr. T. H. Rabbitts), and pRL-CMV (for normalization; obtained from Dr. T. H. Rabbitts)plasmids were cotransfected in RPMI 1640 medium withoutantibiotics into 7.5 104 COS-7 cells in a white 96-well plate(Cellstar, Greiner Bio-One, Monroe, NC) in a 10:10:10:1 ratio.24 h after transfection, themediumwas replacedwith completeRPMI 1640 medium, and 48 h after transfection, both fireflyand Renilla luminescence was measured using the Dual-GloLuciferase Assay (Promega) reagents according to the manu-facturer’s recommendations. Themean fromduplicate sampleswas taken from five separate experiments. pAD-SV40 andpBD-p53 (Stratagene) plasmids were used for the positive control,and pM1 lacking the coiled coil gene was used as the negativecontrol. A relative response ratio was calculated by first nor-

malizing the individual firefly luminescence to theRenilla lumi-nescence. The negative control was subtracted from the meanof the duplicate experimental values and scaled by dividing bythe difference between the positive and negative controls((Experiment Ctrl)/(Ctrl� Ctrl)).Cell Proliferation and Western Blotting—48 h following

transfection of K562 cells with pEGFP-C1, pEGFP-CC, orpEGFP-CCmut2, trypan blue exclusion was used to determinecell proliferation (cell viability). 3 106 cells were pelleted andresuspended in 600 �l of lysis buffer (62.5 mM Tris-HCl, 2%(w/v) SDS, 10% glycerol, 50 mMDTT, 0.01% (w/v) bromphenolblue). Standard Western blotting procedures were followedusing antibodies to detect the phosphorylated (p-) forms of Bcr-Abl, STAT5, CrkL, as well as �-actin as a loading control. Theprimary antibodies (anti-p-Abl(Tyr-245), 73E5, Cell SignalingTechnology; anti-p-STAT5(Tyr-694), E208, Abcam; anti-p-CrkL(Tyr-207), 3181, Cell Signaling Technology; and anti-ac-tin, mAbcam 8226, Abcam) were detected with anti-mouse(Ab6814, Abcam) or anti-rabbit (7074, Cell Signaling Technol-ogy) HRP-conjugated antibodies before the addition of Chemi-Glo (AlphaInnotech, Cell Biosciences, Santa Clara, CA) chemi-luminescent substrate and detection with a FluorChem FC2imager (AlphaInnotech). The resulting bands were quantifiedthrough densitometry and normalized to the �-actin bands.Colony Forming Assay—24 h following transfection of

pEGFP-C1, pEGFP-CC, or pEGFP-CCmut2, K562 cells wereresuspended in Iscove’s modified Dulbecco’s medium (StemCell Technologies, Vancouver, British Columbia, Canada)with2% FBS, and 1,000 cells were seeded inMethocult H4230meth-ylcellulose medium (Stem Cell Technologies) in the absence ofcytokines. Imatinib mesylate (a kind gift from Novartis) wasadded to 1,000 untransfected K562 cells seeded inMethocult atthe time of seeding. Each group of treated cells was seeded intotwo separate plates. Colony formation was assessed 7 days afterseeding cells by counting colonies in a 200-�m2 area of the plateand calculating the mean number of colonies per treatment.Experiments were replicated at least three times and comparedwith control (cells transfected with pEGFP-C1).Caspase-3/7 Assay—48 h following transfection of pEGFP-

C1, pEGFP-CC, or pEGFP-CCmut2, 3 106 K562 cells werepelleted and resuspended in 50�l of lysis buffer provided in theEnzChek Caspase-3/7 Assay Kit 2 (Invitrogen). Cells were fro-zen at 80 °C and then centrifuged at 5,000 g for 5 min.Lysates were transferred to a black 96-well plate (Cellstar,Greiner Bio-One), and 50 �l of 2 7-amino-4-methylcou-marin-DEVD substrate was added and incubated at room tem-perature for 30 min. Fluorescence was measured with excita-tion at 342 nm and emission at 441 nm on a SpectraMax M2plate reader (Molecular Devices, Sunnyvale, CA).Fluorescence Microscopy and DNA Segmentation—48 h fol-

lowing transfection of pEGFP-C1, pEGFP-CC, or pEGFP-CC-mut2, K562 cells were transferred to 2-well live cell chambers, 1�l of the nuclear stain H33342 was added, and the cells wereincubated at 37 °C for 15 min. Cells were then analyzed with aninverted fluorescence microscope (Olympus IX701F, ScientificInstrument Co., Sunnyvale, CA) with a high quality narrowband GFP filter (excitation, HQ480/20 nm; emission,HQ510/20 nm; beam splitter Q4951p; Chroma Technology

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Corp., Brattleboro, VT). Cells were photographed with anF-view Monochrome charge-coupled device camera using a60 objective and were selected based on EGFP fluorescence.The nuclei of at least 50 transfected cells (EGFP fluorescence)per group were classified as either healthy (round or kidney-shaped) or segmented (punctate) (47, 48), and the percentage ofcells with segmented DNA was calculated.

RESULTS

Computational Modeling—Rational design, molecularmodeling, MD, and free energy simulations were used topredict favorable attributes, specificity, and energetics tofacilitate the choice of coiled coil modifications that stabilizebinding of the mutant coiled coil domain with Bcr-Abl(CCmut2-CC) while destabilizing self-oligomerization(CCmut2-CCmut2). Initial simulations monitored increasesin �-helicity as an indirect correlate for improved free energyof binding of mutant pairs (49, 50) and focused on mutationsto potentially improve salt bridge interactions, increase sta-bility through formation of a disulfide bond, improve helicityin the backbone through alanine mutations to position “f,”and destabilize mutations to improve specificity.Alaninemutations in the peptide backbone of theC-terminal

coiled coil region were designed at residues Gln-33, Gln-47,Phe-54, and Thr-61 to increase the �-helicity (49–51). Com-parisons of the helicity as measured by circular dichroism andsecondary structure between the homodimer (CCmut-CCmut)

and heterodimer (CCmut-CC) suggested that this design actu-ally decreases the helicity and shows poor specificity for theheterodimer over the homodimer (see Table 1, row 3, CD andsecondary structure). Visualization of the coiled coil monomerstructure suggests that the designed alanine mutations havedisrupted local intrahelical hydrogen bonds, which may affectsecondary structure and protein folding.Cys-38 exists as an unbound free thiol in the native protein,

and its close proximity to position 52might allow the formationof a disulfide bond that could further stabilize the structure. Anengineered disulfide was modeled by incorporating a cysteineresidue at position 52. Visualization of the heterodimer andanalysis of the structure helicity (see Table 1, row 4, CD) sug-gests that the geometry of the disulfide is not ideal and intro-duces structural disturbances.Three point mutations were designed in the wild-type mon-

omer to improve binding to the oncoprotein: S41R, E48R, andQ60E. When these three designed point mutations were mod-eled, helicity of the homodimer and heterodimer mutantsexceeded the wild-type dimer (see Table 1, compare row 1 withrow 2), suggesting improved binding due to more favorableelectrostatic interactions. The improved helicity of the mutanthomodimer over the Bcr-Abl wild-type dimer, however, mayresult in decreased specificity for the heterodimer form. Desta-bilizing pointmutations to improve the heterodimer specificityincluded designed aspartatemutations in the hydrophobic core

TABLE 1Overview of comparative helicity calculations for various mutations of Bcr coiled coil domainThe helicity of simulated coiled coil dimers using the calculated circular dichroism of the peptide, the percentage of the residue that is defined as �-helical according to �and � backbone dihedral torsions (using the DSSP method), and the percentage of �-helical specific hydrogen bonds (hb) (�3.5 Å) formed between i and i � 4 residues atpicosecond intervals over the final 10 ns of MD relative to the total number of potential interactions (the total number of residues minus 4) are shown. Row 6 contains theonly set of mutations tested that indicated a more favorable homodimer than the heterodimer and was termed CCmut1 and used in subsequentMM-PBSA experiments asa negative control. Row 8 contains the set of mutations found to exhibit the optimal reduced homo-oligomerization paired with improved hetero-oligomerization and wastermedCCmut2 for subsequentMM-PBSAand thermodynamic integration studies aswell as in vitro experiments. The bold values distinguish the experimental values fromthe standard deviations. The italicized mutations are CCmut1 and CCmut2, which were modeled further and then experimentally tested.

MutationsCircular dichroism Secondary structure Hydrogen bonds

�� 222 S.D. Helicity S.D. i, i � 4 hb S.D.

% %(1) None (wild type)Homodimer �20,943 858 66.6 1.7 56.6 2.7

(2) S41R,E48R,Q60EHeterodimer �21,580 672 70.0 2.0 56.9 2.8Homodimer �20,961 678 66.5 2.9 59.4 2.7

(3) Q33A,Q47A,F54A,T61AHeterodimer �20,884 477 66.3 2.5 60.6 2.5Homodimer �20,787 760 65.1 2.2 56.4 3.2

(4) E52CHeterodimer �20,521 796 67.8 2.2 55.9 2.7

(5) S41R,L45D,E48R,Q60EHeterodimer �20,330 1,329 66.8 1.9 52.9 3.0Homodimer �19,398 679 66.2 2.0 56.5 2.4

(6) S41R,L45D,E48R,V49D,Q60E (CCmut1)Heterodimer �19,696 765 67.8 2.0 56.9 2.7Homodimer �20,352 671 69.1 2.5 59.2 2.8

(7) C38A,S41R,E48R,Q60EHeterodimer �21,493 90 70.0 2.4 61.5 2.5Homodimer �21,463 265 70.0 2.6 61.4 2.5

(8) C38A,S41R,L45D,E48R,Q60E (CCmut2)Heterodimer �21,186 695 67.7 2.4 59.1 2.8Homodimer �17,964 507 59.3 2.6 50.7 2.4

(9) C38A,S41R,L45D,E48R,V49D,Q60EHeterodimer �19,651 456 64.2 2.5 54.0 2.5Homodimer �16,770 312 64.2 1.8 50.4 3.3

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(L45D and L49D) and were evaluated as both single and doublemutations. Both mutations together decreased the secondarystructure (and stability) of the heterodimer (see Table 1, rows 6and 9), whereas L45D maintained heterodimer specificity (seeTable 1, row 5). The final mutation converted an exposed thiolgroup (Cys-38) to alanine and, in conjunction with the fourprior mutations, stabilized the heterodimer over both themutant homodimer and the wild-type dimer (see Table 1, com-pare row 1 with row 8). Thus, the optimal mutant coiled coildomain (termed CCmut2) contains C38A, S41R, L45D, E48R,and Q60E mutations. Specificity as measured by helicity sug-gests that themutant homodimer (CCmut2-CCmut2) is signif-icantly less stable than the wild type (CC-CC) and the het-erodimer (CC-CCmut2).MM-PBSA and thermodynamic integration energy calcula-

tion methodologies (the latter are more quantitative and accu-rate) were subsequently used to analyze the two sets of muta-tions. Both approaches validated the previous indicationssuggesting that CCmut2 exhibits improved heterodimer stabil-ity compared with the wild-type oligomerization (see Table 2).Furthermore, CCmut2 was shown to have reduced homo-olig-omerization. Together, MM-PBSA and thermodynamic inte-gration energy calculations suggest improved binding and spec-ificity of CCmut2.In Vitro Experiments Validate Design—To substantiate the

computational circular dichroism calculations, wavelengthscanswere performed on protein solutions of CCmut2, CC, anda mixture of CCmut2 and CC. As seen in Fig. 2A, all threeprotein solutions produced the typical pattern characteristic of�-helices with similar helicity. Given the relatively small num-ber of mutations that distinguish CCmut2 from wild-type CCand because the mutations are designed to alter oligomeriza-tion while retaining helicity, it is reasonable to expect no majordifferences in their experimentallymeasured helicity.However,themodifications do clearly destabilize themutant homodimeras made apparent in the thermal denaturation of the proteins(Fig. 2B). Although CC demonstrated a melting temperature(Tm) consistent with previous reports of 83 °C (23), a largedecrease in Tm was found for the CCmut2 (Tm � 63 °C) (Fig.2B), confirming the decreased stability of the mutant homo-oligomer.When CCmut2 wasmixed with CC in a 1:1 ratio, twodistinct melting transitions were evident (Fig. 2B, black trian-

gles); however, a third Tm was not readily apparent. This sug-gests that the CC-CCmut2 heterodimer is either isoenergeticwith one of the two homodimers or that it simply did not form.To further assess the formation of the heterodimer, a 3:1(CCmut2-CC) ratio was studied in a mixing cell cuvette (Fig.2C) both before and after mixing. The mixing shifted the curvemost predominantly at lower temperatures and only slightly athigher temperatures, suggesting the formation of a new species(as seen in Fig. 2C). The shift in the curve at lower temperaturescan be accounted for by the decreased CCmut2 concentrationdue to formation of heterodimers. The small difference in thecurves at higher temperatures suggests the formation of hetero-oligomers that are nearly isoenergetic or slightly less stable thanthe CC homo-oligomers. Consistent with the computational

TABLE 2MM-PBSA and thermodynamic relative free energy of bindingresults (in kcal/mol)MM-PBSA results were found by subtracting the absolute free energies of theunbound monomers from the calculated free energy of coiled coil dimer usingseparate MD trajectories. Thermodynamic integration calculations followed thescheme described in supplemental Fig. S2 using intermediate coiled coil dimers tobuild a consistent transition from the wild-type coiled coil dimer to the CCmut2dimers. Results from both calculations are reported relative to the wild-type coiledcoil dimer (CC-CC). ND, not determined.

DimerMM-PBSA Thermodynamic integration

��Gbinding S.E. ��Gbinding S.D.

kcal/mol kcal/molCC-CC 0.0 3.2 0.0 0.0CC-CCmut1 11.7 3.0 ND NDCCmut1-CCmut1 25.5 0.1 ND NDCC-CCmut2 1.3 3.1 1.1 0.5CCmut2-CCmut2 11.4 3.0 3.2 0.8

FIGURE 2. Circular dichroism wavelength scans and thermal denatur-ation. A, �-helices exhibit a characteristic double absorption minimum at�208 and 222 nm. Gray circles, CC; black squares, CCmut2; black triangles,mixture (Mix). The lines represent the average from three scans. B, thermaldenaturation curves in a 1-cm cuvette. The ratio of CC to CCmut2 used in themixture was 1:1. Gray circles, CC; black squares, CCmut2; black triangles, mix-ture. C, thermal denaturation curves in a mixing cell cuvette using 2.5 �M CCand 7.5 �M CCmut2. Gray circles, separate (premixing); black squares, mixture.deg, degrees.

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modeling, the primary improvement made through thesemutations is in the specificity granted by a less stable mutanthomodimer while retaining the ability to oligomerize with wildtype.The optimally designed mutant coiled coil was created

through site-directed mutagenesis of a plasmid encoding theBcr-Abl coiled coil for cell-based in vitro experiments and fur-ther validation. First, the NTA (46) was used to study the inter-action (Fig. 3A). This assay measures the ability of a nuclearlocalization-inducible PS fused to one form of the coiled coildomain to alter the nuclear localization of another form of thecoiled coil domain. Essentially, the interaction between thecoiled coil domains is indicated by an increase in fluorescencein the nucleus. The high level of nuclear translocation resultingfrom the mutant coiled coil domain (Fig. 3A,middle column) islikely to stem from both the reduced homo-oligomerizationand the improved binding to the wild-type coiled coil domain.To specifically address whether the designedmutations limitedthe homo-oligomerization, the interaction between twomutant coiled coil domains (CCmut2-CCmut2) was assayedand found to be indistinguishable from the negative control(not included in the graph). These same interactions were alsostudied in a mammalian two-hybrid assay to further validatethe results. Similar to the NTA results, the greatest binding wasfound between the mutant and wild-type coiled coil domains(CCmut2-CC; Fig. 3B, second column), and the homo-olig-omerization between two mutants (CCmut2-CCmut2; Fig. 3B,third column) was not statistically distinguishable from thenegative control (not included in the graph). Together, theNTA and two-hybrid results indicate that the proposed muta-tions reduced the homo-oligomerization of the mutant coiledcoil domain and improved the binding to the wild-type coiledcoil domain.Next, the oligomeric disruption of Bcr-Abl was indirectly

measured through assaying the activity of Bcr-Abl. As the olig-omeric state of Bcr-Abl is correlated to its activity, if the olig-

omerization is disrupted it will cause a reduction in Bcr-Ablactivity (decrease in phosphorylation). After transfecting eitherthe wild-type or mutant coiled coil domain into K562 cells,Western blotting with an antibody that specifically recognizesthe phosphorylated form of Bcr-Abl was used to determine theactivity. Under identical experimental conditions, thewild-typecoiled coil domain hadminimal effect on the level of phosphor-ylation of Bcr-Abl (Fig. 4A, third column), whereas the mutantcoiled coil domain reduced the phosphorylation level to 35%(Fig. 4A, last column). Furthermore, the phosphorylation of twoproteins known to be phosphorylated by Bcr-Abl, STAT5 andCrkL, was also tested. Again, the wild-type coiled coil domainhad minimal effect, whereas the mutant coiled coil domainreduced the phosphorylation of both proteins (Fig. 4B, thirdand fourth columns). The decreased phosphorylation of Bcr-Abl suggests that the mutant coiled coil domain is capable ofinteracting with the endogenous Bcr-Abl and not just the iso-lated coiled coil domain as used in the previous NTA and two-hybridexperiments.Moreover, thedecreased levelofphosphor-ylation provides insight into the oligomeric disruption andinhibitory potential of the mutant coiled coil domain.Inhibition of Bcr-Abl through oligomeric disruption (as

with inhibition through tyrosine kinase inhibitors) shouldrelieve the up-regulation of signaling pathways resulting inmisregulated cell proliferation. The effect of the coiled coildomains on cell proliferation was measured through cellcounts with trypan blue exclusion. Although the wild-typecoiled coil domain demonstrated a slight effect on the num-ber of proliferating cells, the mutant coiled coil domain wasmost effective at decreasing the number of proliferating cells(Fig. 5A). Furthermore, the effect on proliferation was mea-sured via a colony forming assay, and again themutant coiledcoil domain was found to cause the greatest reduction in cellproliferation (Fig. 5B, column 2) similar to that seen withimatinib (Fig. 5B, column 3).

FIGURE 3. Binding of homo- and heterodimers tested through NTA and mammalian two-hybrid assay. A, figure was modified from Dixon and Lim (46).The NTA measures the ability of a nuclear localization-inducible PS fused to a protein of interest to cause a second interacting protein to be translocated intothe nucleus and is monitored through fluorescence microscopy (fused to EGFP and DsRed, respectively). The assay was performed in COS-7 cells that do notcontain Bcr-Abl. Each experiment was performed in triplicate with at least eight cells analyzed per experiment. Statistical significance was determined usingone-way ANOVA with Tukey’s post-test. *, p � 0.01; **, p � 0.001 compared with control (pDsRed2-N1/EGFP-PS-CC; not included in the graph). B, mammaliantwo-hybrid assay in COS-7 cells. Statistical significance was determined using one-way ANOVA with Tukey’s post-test. *, p � 0.01 compared with CC-CCinteraction (n � 5). Error bars represent � S.D.

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As CML cells become dependent on the signaling path-ways up-regulated by Bcr-Abl, the inhibition of Bcr-Abl andreprieve of that signaling should also induce apoptosis. Asone indication of the ability of the coiled coil constructs toinduce apoptosis, the activity of caspase-3/7 was measuredin a fluorometric assay. In a trend similar to that found in allprevious experiments, the mutant coiled coil domain againproduced the greatest result and was the only construct ableto induce the activation of caspase activity at a statisticallysignificant level (Fig. 5C, third column). As expected, similarexperiments with CCmut2 in cells that do not express Bcr-Abl (1471.1 and COS-7) did not show an increase in caspaseactivity (data not shown).Finally, as a measure of late stage apoptosis, DNA segmenta-

tion (of K562 nuclei) was measured (47, 48). Cells transfectedwith CCmut2 revealed segmented nuclei, a hallmark of apopto-sis as shown in Fig. 6 (column 3, arrows). CC- and control(EGFP)-transfected cells had healthy (round) nuclei. The per-centage of CCmut2-transfected cells demonstrating apoptosis

was 29.4% compared with 4.29% for CC and 0.75% for EGFPcontrol. Additionally, phase-contrast images (Fig. 6, column 1)demonstrated morphological changes indicative of apoptosis,including zeotic blebbing, cell shrinkage, and cell fragmenta-tion (43, 48). Finally, in a control cell line (1471.1 cells),minimalDNA segmentation (less than 2%) was observed after EGFP,CC, or CCmut2 was transfected (data not shown). The inhibi-tion of cell proliferation and the induction of apoptosis illus-trate the therapeutic potential of oligomeric disruption throughthis modified coiled coil.

DISCUSSION

Rational design, molecular modeling, MD simulation, andfree energy analysis identified modifications to the Bcr-Ablcoiled coil to improve interaction with Bcr-Abl while alsoreducing mutant homodimer (CCmut2-CCmut2) interactions.The optimal set of mutations served as the lead, reducing theneed to test an overwhelming number of possible mutations orcombination of mutations in vitro. The in vitro experiments

FIGURE 4. Representative images of Western blots to detect phosphorylated form of Bcr-Abl (A) and two substrates of Bcr-Abl, STAT5 and CrkL (B). Thephosphorylation of Bcr-Abl is indicative of the tyrosine kinase activity and is shown to be decreased by the addition of CCmut2 (percentage of p-Bcr-Abl fromuntreated K562 cells is indicated graphically). The proteins STAT5 and CrkL are also phosphorylated when Bcr-Abl is active and are secondary indicators of theBcr-Abl activity. Western blotting followed by densitometry was replicated three times on lysates from three separate transfections. The level of p-Bcr-Abl, asa percentage of the untreated cells, is shown graphically in A, and the level of p-STAT5 and p-CrkL (�S.D.) is indicated above the representative images.Statistical significance was determined using one-way ANOVA with Tukey’s post-test (n � 3). *, p � 0.05; **, p � 0.01 compared with cells transfected withEGFP. Error bars represent � S.D.

FIGURE 5. Inhibition of Bcr-Abl through expression of CCmut2 results in decreased proliferation of K562 cells and activation of apoptosis. A, prolifer-ation of K562 cells as determined by cell counts with trypan blue exclusion. B, proliferation of K562 cells as determined by colony forming assays. IM, imatinibmesylate. C, induction of apoptosis as measured through activation of caspase-3/7. For A–C, statistical significance was determined using one-way ANOVA withTukey’s post-test. *, p � 0.01; **, p � 0.001 compared with control (cells transfected with pEGFP-C1). Error bars represent � S.D.

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performed with this construct confirmed the computationalresults and demonstrated that this designed mutant coiled coilhas an enhanced capability to oligomerize with Bcr-Abl. Thedesign incorporated charge-charge repulsions between twomutant coiled coil domains to reduce the homo-oligomeriza-tion, thereby making the mutant more available for interactionwith the target, Bcr-Abl. Residues with the potential to formadditional favorable electrostatic interactions with Bcr-Ablwere also introduced to increase the binding affinity betweenthemutant andBcr-Abl. Although further structural character-ization could confirm that the hypothesized interactions areindeed occurring, both the computational modeling and invitro experiments strongly indicate that the modifications tothe coiled coil domain lead to a more specific, better bindingcoiled coil partner for Bcr-Abl.Although an isolated coiled coil domain from Bcr-Abl

should in principal oligomerize with Bcr-Abl, the isolatedcoiled coil domain also has the ability to form homo-oligom-ers. Given that an isolated coiled coil domain is smaller,there is likely less entropic penalty for formation, and there-fore it should be less effective as a therapeutic due to thedecreased effective concentration of the monomer and needfor dissociation of the dimer for activity. Our approach to amore potent therapeutic involved the design of a coiled coildomain with reduced ability to self-oligomerize while alsoexhibiting enhanced oligomerization with the target. Thisgoal was confirmed through both computational modelingand in vitro experiments. Alternative treatments for CMLare still needed because of the inability to eliminate CMLstem cells, resistance to small molecule inhibitors, and inef-fective treatment of advanced stages of the disease (25,52–55). Given the importance the coiled coil domain has inthe regulation of Bcr-Abl activity, it has long been hypothe-sized that this domain could be used therapeutically. Thismodified Bcr-Abl coiled coil domain has a heightened ability

to inhibit the oncogenicity of Bcr-Abl and warrants furtherexploration as an alternative approach to treat CML.

Acknowledgments—We acknowledge the use of the DNA/PeptideCore (NCI Cancer Center Support Grant P30 CA042014, HuntsmanCancer Institute) and computer time from the Center for High Per-formance Computing at the University of Utah. We thank Dr. DebbieEckert for technical assistance in protein purification and JonathanConstance, Rian Davis, Mohanad Mossalam, Matthew Weinstock,and Dr. Michael Kay for scientific discussions.

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Disruption of Bcr-Abl Coiled Coil Oligomerization by Design

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