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Disruption of Bcr-Abl Coiled Coil Oligomerization by Design*

  • Andrew S. Dixon
    Affiliations
    Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108
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  • Scott S. Pendley
    Affiliations
    Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108
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  • Benjamin J. Bruno
    Affiliations
    Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108
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  • David W. Woessner
    Affiliations
    Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108
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  • Adrian A. Shimpi
    Affiliations
    Juan Diego Catholic High School, Draper, Utah 84020
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  • Thomas E. Cheatham III
    Correspondence
    To whom all other correspondence should be addressed: Dept. of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, 421 Wakara Way, Rm. 318, Salt Lake City, UT 84108. Fax: 801-585-3614.
    Affiliations
    Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108

    Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108
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  • Carol S. Lim
    Correspondence
    To whom computational correspondence should be addressed.
    Affiliations
    Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants CA129528 (to C. S. L.) and GM079383 (to T. E. C.). This work was also supported by an American Foundation for Pharmaceutical Education predoctoral fellowship (to A. S. D), Graduate Research Fellowship, University of Utah (to A. S. D.) the ALSAM Foundation (to A. A. S), and National Science Foundation Grant TG-MCA01S027 (to T. E. C.; for computer time).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
Open AccessPublished:June 09, 2011DOI:https://doi.org/10.1074/jbc.M111.264903
      Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

      Introduction

      Coiled coil domains are ubiquitous protein structural motifs found in ∼10% of all eukaryotic proteins (
      • Taylor C.M.
      • Keating A.E.
      ). Characterized by a heptad repeat (sequence of seven amino acids) and association of two or more α-helices, coiled coils provide oligomerization capabilities (both homo- and hetero-oligomerization) useful for structural scaffolding and protein recognition. Coiled coil domains are critical for regulating many processes involved in the pathogenesis of various diseases (
      • Strauss H.M.
      • Keller S.
      ). Thus, rationally designed coiled coils can be used as therapeutics by interfering with the activity of a pathogenic protein through oligomeric disruption. One example is enfuvirtide (Fuzeon®), a fusion inhibitor that disrupts the helix bundle formation necessary for HIV-1 viral entry (
      • Matthews T.
      • Salgo M.
      • Greenberg M.
      • Chung J.
      • DeMasi R.
      • Bolognesi D.
      ), spawning the next generation of rationally designed fusion inhibitors (
      • Dwyer J.J.
      • Wilson K.L.
      • Davison D.K.
      • Freel S.A.
      • Seedorff J.E.
      • Wring S.A.
      • Tvermoes N.A.
      • Matthews T.J.
      • Greenberg M.L.
      • Delmedico M.K.
      ,
      • Otaka A.
      • Nakamura M.
      • Nameki D.
      • Kodama E.
      • Uchiyama S.
      • Nakamura S.
      • Nakano H.
      • Tamamura H.
      • Kobayashi Y.
      • Matsuoka M.
      • Fujii N.
      ,
      • Yan Z.
      • Tripet B.
      • Hodges R.S.
      ,
      • Eckert D.M.
      • Malashkevich V.N.
      • Hong L.H.
      • Carr P.A.
      • Kim P.S.
      ,
      • Sia S.K.
      • Carr P.A.
      • Cochran A.G.
      • Malashkevich V.N.
      • Kim P.S.
      ). The coiled coil reported here was designed to bind to the target (Bcr-Abl) better than the target protein binds to itself while exhibiting minimal homo-oligomerization.
      Coiled coils are very well characterized, and the high correlation between their sequence and structure (
      • Parry D.A.
      • Fraser R.D.
      • Squire J.M.
      ) is advantageous for rational design. Coiled coil oligomerization involves hydrophobic interactions, salt bridge formation, and helicity. In the heptad repeat, hydrophobic packing at the “a” and “d” positions (see Fig. 1, green residues) helps drive association with further stability provided by the residues at positions “g” and “e,” which are commonly charged and interact to form salt bridges (see Fig. 1, red and blue residues). Because charged residues are routinely found at the g and e positions, mutating these residues in a rational manner to add salt bridges to favor formation of hetero-oligomers (
      • Kammerer R.A.
      • Jaravine V.A.
      • Frank S.
      • Schulthess T.
      • Landwehr R.
      • Lustig A.
      • Garcia-Echeverria C.
      • Alexandrescu A.T.
      • Engel J.
      • Steinmetz M.O.
      ,
      • Spek E.J.
      • Bui A.H.
      • Lu M.
      • Kallenbach N.R.
      ,
      • Marqusee S.
      • Baldwin R.L.
      ) and charge-charge repulsions to reduce the formation of homo-oligomers (
      • Ryan S.J.
      • Kennan A.J.
      ,
      • Gurnon D.G.
      • Whitaker J.A.
      • Oakley M.G.
      ) can change the affinity and specificity of the coiled coil dimer.
      Figure thumbnail gr1
      FIGURE 1Ribbon diagrams (with corresponding helical wheel diagram below) of wild-type homodimer (A), wild-type-CCmut2 heterodimer (B), and CCmut2-CCmut2 homodimer (C). Gray ribbons (ribbon diagrams) or dots (helical wheel diagrams) represent the wild-type coiled coil domain, and cyan represents 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 as red (acidic), blue (basic), green (hydrophobic), yellow (serine), or black (glutamine) spheres (ribbon diagrams) or font (helical wheel diagrams). Dotted lines indicate possible ionic interactions, and solid lines indicate charge-charge repulsions. Ribbon diagrams were generated with UCSF Chimera starting with the Bcr coiled coil domain crystal structure (Protein Data Bank code 1K1F).
      Bcr-Abl exists primarily as a tetramer (more specifically as a dimer of dimers), facilitating the trans-autophosphorylation necessary to activate the tyrosine kinase domain (
      • Zhang X.
      • Subrahmanyam R.
      • Wong R.
      • Gross A.W.
      • Ren R.
      ,
      • Arlinghaus R.B.
      ). Oligomerization of Bcr-Abl is achieved through a coiled coil domain at the N terminus of the protein. Bcr-Abl constructs lacking the N-terminal coiled coil fail to induce chronic myelogenous leukemia (CML)
      The abbreviations used are: CML
      chronic myelogenous leukemia
      CC
      wild-type coiled coil domain from Bcr-Abl
      CCmut1
      mutant coiled coil domain with S41R, L45D, E48R, V49D, and Q60E mutations
      CCmut2
      mutant coiled coil 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.
      in a murine model (
      • Zhang X.
      • Subrahmanyam R.
      • Wong R.
      • Gross A.W.
      • Ren R.
      ,
      • He Y.
      • Wertheim J.A.
      • Xu L.
      • Miller J.P.
      • Karnell F.G.
      • Choi J.K.
      • Ren R.
      • Pear W.S.
      ), thus setting the stage for oligomeric disruption as a therapy. Proof of concept for using the coiled coil domain to inhibit Bcr-Abl activity has already been demonstrated through retroviral transfection of the wild-type coiled coil domain or addition of the purified protein attached to a cytoplasmic transduction peptide (
      • Mian A.A.
      • Oancea C.
      • Zhao Z.
      • Ottmann O.G.
      • Ruthardt M.
      ,
      • Beissert T.
      • Hundertmark A.
      • Kaburova V.
      • Travaglini L.
      • Mian A.A.
      • Nervi C.
      • Ruthardt M.
      ,
      • Beissert T.
      • Puccetti E.
      • Bianchini A.
      • Güller S.
      • Boehrer S.
      • Hoelzer D.
      • Ottmann O.G.
      • Nervi C.
      • Ruthardt M.
      ,
      • Huang S.F.
      • Liu D.B.
      • Zeng J.M.
      • Xiao Q.
      • Luo M.
      • Zhang W.P.
      • Tao K.
      • Wen J.P.
      • Huang Z.G.
      • Feng W.L.
      ,
      • Guo X.Y.
      • Cuillerot J.M.
      • Wang T.
      • Wu Y.
      • Arlinghaus R.
      • Claxton D.
      • Bachier C.
      • Greenberger J.
      • Colombowala I.
      • Deisseroth A.B.
      ). To further this approach, we designed and tested a mutant coiled coil (CCmut2) using both computational and in vitro experiments. This CCmut2 disfavors self-oligomerization (CCmut2:CCmut2) yet appears to bind more tightly to the target Bcr-Abl coiled coil domain (CCmut2:Bcr-Abl) than the 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 α-helices (α1 (residues 5–15) and α2 (residues 28–67)) that form an N-shaped configuration with two parallel helices connected by a short linker (see Fig. 1A) (
      • Zhao X.
      • Ghaffari S.
      • Lodish H.
      • Malashkevich V.N.
      • Kim P.S.
      ). Upon dimerization, the resulting coiled coil has an antiparallel orientation with α2 at the core and α1 latching onto the backside of the opposing α2 helix (domain swapping) (
      • Taylor C.M.
      • Keating A.E.
      ,
      • Zhao X.
      • Ghaffari S.
      • Lodish H.
      • Malashkevich V.N.
      • Kim P.S.
      ). The majority of the dimer interface is composed of the classic knobs-in-holes type hydrophobic interactions from residues at the a and d positions (
      • Crick F.H.
      ). Further stabilization comes from four interchain salt bridges between residues in the α2 helices (Fig. 1A) as well as packing of aromatic residues from the α1 helix and opposing α2 helix. As seen in Fig. 1A, there are two uncharged residues (Ser-41 and Gln-60) that are in the appropriate position for the formation of salt bridges with two charged residues (Glu-32 and Glu-48). Thus, mutation of Ser-41 and Gln-60 to positively charged amino acids has the potential to provide two additional salt bridges with Bcr-Abl (Fig. 1B) and thus enhance binding. However, although this provides more salt bridges in the hetero-oligomer, these mutations alone are undesirable as they allow the formation of a greater number of salt bridges in the homo-oligomer. To reduce homo-oligomerization in the mutant coiled coil, residues proximal to charged residues on the opposing helix were considered as candidates for mutation to introduce charge-charge repulsion (
      • Ryan S.J.
      • Kennan A.J.
      ,
      • Gurnon D.G.
      • Whitaker J.A.
      • Oakley M.G.
      ). Leu-45 and Glu-48 were identified as two such residues (Fig. 1C). In addition, previous reports have incorporated a C38A mutation primarily for crystallization purposes (
      • Taylor C.M.
      • Keating A.E.
      ,
      • Zhao X.
      • Ghaffari S.
      • Lodish H.
      • Malashkevich V.N.
      • Kim P.S.
      ), and this mutation was also studied. Putative mutations were investigated first through molecular modeling and state-of-the art biomolecular simulation and free energy analysis to ascertain the impact on the coiled coil structure and stability. Such in silico methods provide a means to efficiently (and inexpensively) assess the influence of mutation. The resulting optimized mutant coiled coil (CCmut2) contains five mutations (C38A, S41R, L45D, E48R, and Q60E) and was further assessed in vitro. Taken together, these results demonstrate the effectiveness of the mutant coiled coil domain and, importantly, further illustrate the ability to rationally modify an existing coiled coil domain to improve therapeutic efficacy.

      DISCUSSION

      Rational design, molecular modeling, MD simulation, and free energy analysis identified modifications to the Bcr-Abl coiled coil to improve interaction with Bcr-Abl while also reducing mutant homodimer (CCmut2-CCmut2) interactions. The optimal set of mutations served as the lead, reducing the need to test an overwhelming number of possible mutations or combination of mutations in vitro. The in vitro experiments performed with this construct confirmed the computational results and demonstrated that this designed mutant coiled coil has an enhanced capability to oligomerize with Bcr-Abl. The design incorporated charge-charge repulsions between two mutant coiled coil domains to reduce the homo-oligomerization, thereby making the mutant more available for interaction with the target, Bcr-Abl. Residues with the potential to form additional favorable electrostatic interactions with Bcr-Abl were also introduced to increase the binding affinity between the mutant and Bcr-Abl. Although further structural characterization could confirm that the hypothesized interactions are indeed occurring, both the computational modeling and in vitro experiments strongly indicate that the modifications to the coiled coil domain lead to a more specific, better binding coiled coil partner for Bcr-Abl.
      Although an isolated coiled coil domain from Bcr-Abl should in principal oligomerize with Bcr-Abl, the isolated coiled coil domain also has the ability to form homo-oligomers. Given that an isolated coiled coil domain is smaller, there is likely less entropic penalty for formation, and therefore it should be less effective as a therapeutic due to the decreased effective concentration of the monomer and need for dissociation of the dimer for activity. Our approach to a more potent therapeutic involved the design of a coiled coil domain with reduced ability to self-oligomerize while also exhibiting enhanced oligomerization with the target. This goal was confirmed through both computational modeling and in vitro experiments. Alternative treatments for CML are still needed because of the inability to eliminate CML stem cells, resistance to small molecule inhibitors, and ineffective treatment of advanced stages of the disease (
      • Martin M.G.
      • Dipersio J.F.
      • Uy G.L.
      ,
      • Santos F.P.
      • Ravandi F.
      ,
      • Kantarjian H.M.
      • Cortes J.
      • La Rosée P.
      • Hochhaus A.
      ,
      • Jamieson C.H.
      ,
      • Janssen J.J.
      • Schuurhuis G.J.
      • Terwijn M.
      • Ossenkoppele G.J.
      ). Given the importance the coiled coil domain has in the regulation of Bcr-Abl activity, it has long been hypothesized that this domain could be used therapeutically. This modified Bcr-Abl coiled coil domain has a heightened ability to inhibit the oncogenicity of Bcr-Abl and warrants further exploration as an alternative approach to treat CML.

      Acknowledgments

      We acknowledge the use of the DNA/Peptide Core (NCI Cancer Center Support Grant P30 CA042014, Huntsman Cancer Institute) and computer time from the Center for High Performance Computing at the University of Utah. We thank Dr. Debbie Eckert for technical assistance in protein purification and Jonathan Constance, Rian Davis, Mohanad Mossalam, Matthew Weinstock, and Dr. Michael Kay for scientific discussions.

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