Modeling of Molecular Interaction between

Apoptin, BCR-Abl and CrkL - An Alternative

Approach to Conventional Rational Drug
Design

Soumya Panigrahi, Joerg Stetefeld, Jaganmohan R. Jangamreddy, Soma Mandal,

Sanat K. Mandal and Marek Jan Los

Linkping University Post Print

N.B.: When citing this work, cite the original article.

Original Publication:

Soumya Panigrahi, Joerg Stetefeld, Jaganmohan R. Jangamreddy, Soma Mandal, Sanat K.

Mandal and Marek Jan Los, Modeling of Molecular Interaction between Apoptin, BCR-Abl
and CrkL - An Alternative Approach to Conventional Rational Drug Design, 2012, PLoS
ONE, (7), 1, 6-20.
http://dx.doi.org/10.1371/journal.pone.0028395
Copyright: Public Library of Science
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Postprint available at: Linkping University Electronic Press

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Modeling of Molecular Interaction between Apoptin,
BCR-Abl and CrkL - An Alternative Approach to
Conventional Rational Drug Design
Soumya Panigrahi1, Jorg Stetefeld2, Jaganmohan R. Jangamreddy7, Soma Mandal3, Sanat K. Mandal4,5,
Marek Los6,7*
1 Department of Molecular Cardiology, Lerner Research Institute/NB-50, Cleveland, Ohio, United States of America, 2 Department of Chemistry, University of Manitoba,
Winnipeg, Canada, 3 Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada, 4 Faculty of Medicine, Memorial University of Newfoundland, St. Johns,
Newfoundland, Canada, 5 College of the North Atlantic, Clarenville, Newfoundland, Canada, 6 BioApplications Enterprises, Winnipeg, Manitoba, Canada, 7 Department of
Clinical and Experimental Medicine (IKE) and Integrative Regenerative Medicine Center (IGEN), Linkoping University, Linkoping, Sweden

Abstract
In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its
interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the
newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous
leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains
cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact
with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and
survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these
domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like
sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and
its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the
mode of apoptins action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-
dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-
Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions
that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective
toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with
greater selectivity and potency.

Citation: Panigrahi S, Stetefeld J, Jangamreddy JR, Mandal S, Mandal SK (2012) Modeling of Molecular Interaction between Apoptin, BCR-Abl and CrkL - An
Alternative Approach to Conventional Rational Drug Design. PLoS ONE 7(1): e28395. doi:10.1371/journal.pone.0028395
Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America
Received October 12, 2011; Accepted November 7, 2011; Published January 10, 2012
Copyright: 2012 Panigrahi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Memorial University and College of the North Atlantic, Clarenville Campus, are gratefully acknowledged for their support (SKM). JS thankfully
acknowledges the support by the Canada Research Chair program. ML kindly acknowledges the core/startup support from Linkoping University, from Integrative
Regenerative Medicine Center (IGEN, a non-for profit research structure within the university), and from Cancerfonden (CAN 2011/521). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: ML is employed by BioApplications Enterprises. This does not alter the authors adherence to all the PLoS ONE policies on sharing data
and materials.
* E-mail: [email protected]

Introduction However, failure of response occurs in advanced-stage CML

patients due to drug resistance caused frequently by mutations
Aberrant expression of the Bcr-Abl oncogene is found with the within- or in the proximity to Bcr-Abls ATP-binding pocket.
frequency of 695% of CML cases, and sporadically in other Other types of changes have also been documented. For example,
malignancies [1,2]. Thus for therapeutic applications, especially CML stem cells obtained from some patients with imatinib
for CML treatment, Bcr-Abl is an attractive target for rational resistance, down-regulation the expression of the tumor suppressor
drug design, although so far, only its tyrosine kinase domain has PTEN could be detected [4]. Although, the second-generation
been utilized. Bcr-Abl oncoprotein, contains a number of distinct tyrosine kinase inhibitors namely, dasatinib, nilotinib or bosutinib
domains such as SH3, SH2, kinase domains, DNA binding are effective on most of the Bcr-Abl mutations, some p-loop
domains, actin-binding domains, nuclear localization signals, mutations or T315I substitution provide a very difficult resistance
nuclear export signal, and four proline-rich motifs that function to most of the Bcr-Abl kinase inhibitors currently in use [2,5,6].
as binding sites for the adaptor proteins such as, Grb2 and CrkL Other therapeutic strategies besides small molecule approach
[2]. Several potent inhibitors have been developed and studied include the use of monoclonal antibodies [7].
extensively [3]. Imatinib is currently widely used for the treatment Current understanding of resistance mechanisms include the
of CML patients. Most chronic phase CML patients treated with ability of cancer cells to remove drugs (drug efflux) by the different
imatinib as first-line therapy, initially maintain excellent response. transporters such as MDR1 (multidrug resistance protein 1) or P-

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 Apoptin Structure

glycoprotein, lack of bioavailability of drugs, and inhibition of amino acid sequences (target) on the basis of known 3D structure
transporter molecules such as SLC22A1 (solute carrier family 22 of related family members (templates). The low resolution models
member 1), responsible for drug transport into the cell (drug obtained by homology modeling provide essential information of
influx), or mutations altering the interaction of drug with its target. the spatial arrangement of important groups of residues.
These mechanisms however do not fully explain drug-resistance Here we report for the first time, a simulated 3D model of
observed in all instances. One of the possible reasons could be the apoptin generated by using homology modeling as a backup
protein dynamics due to drug-protein binding (protein dynamics alternative to the conventional, crystallography- or NMR-based
due to folding, change in shape and size). It is known that the methods. We then use the calculated structural coordinates of
kinase domain of Bcr-Abl is negatively regulated, in normal apoptin to study its interaction with the 3D structure of CML-
situation, by cooperative combination of the SH3 and SH2 associated oncoprotein Bcr-Abl. Furthermore, we biochemically
domain by internally engaging the SH2 domain [8,9]. Generally, confirmed the accuracy of at least some elements of the model by
SH3 domains serve as modules that mediate protein-protein showing that the modeled interaction between apoptin and Bcr-
associations along with SH2 domains and thus regulate cytoplas- Abl indeed physically occurs between both proteins. We also
mic signaling. SH2 domains play important roles in (i) in cellular examined the pathways and interacting network from a global
communication, (ii) in a variety signal transduction pathways, and perspective and experimentally validated some of the important
(iii) in recognition of tyrosine-phosphorylated sites respectively. But molecules.
inappropriate communication or misreading of the phosphorylat-
ed site could lead to undesirable activation of pathways [10,11]. Results
Negative regulation of Bcr-Abl by blocking these domains by
apoptin-inspired small molecule to control its oncogenic role Apoptin is toxic to both Bcr-Abl positive and -negative
would be an attractive approach, as compared to conventional cells, and it inhibits Bcr-Abl phosphorylation/activation
targeted drug design [12]. In this study, we present a possible The known domains of apoptin were designed and presented
alternative approach of inhibition of Bcr-Abl through surface (Fig. 1A) to understand its cytotoxicity in relation to its various
interaction of SH3 domain by the apoptin molecule rather than domains. As seen by the MTT assay performed on the
binding to a narrowly-defined domain. representative murine 32Dp210 cell line, Tat-apoptin efficiently
Apoptin has gained significant attention in recent years, both as killed those cells as compared to control (Tat-GFP, Fig. 1B).
a lead for the development of cancer-specific therapeutics, and also Furthermore, apoptins toxicity favorably compared to imatinib.
for its potential use as an indicator of cellular transformation Evaluation of percentage cell survival was followed by the
processes. Apoptin is a 13.6 kD viral protein encoded by the VP3 examination of the phosphorylation status of Bcr-Abl in the
gene of Chicken Anemia Virus and is composed of 121 amino human leukemia cell line, K562 and the mouse cell line 32Dp210.
acids [13,14]. It induces apoptosis independently of death receptor Tat-apoptin markedly inhibited phosphorylation of Bcr-Abl in
pathways in a broad range of transformed and cancer cells. both cell lines. Inhibition of phosphorylation of Bcr-Abl was
Apoptin localizes in the nucleus in cancer cells, however in non- evaluated by Western blotting (Fig. 1C) and quantified (Fig. 1D).
transformed or primary cells it is localized to the cytoplasm
[15,16,17]. The cellular localization of apoptin is influenced by its Identification of the SH3 binding domain of apoptin
phosphorylation status at theronine-108. Phosphorylated T-108 SH3 Hunter software, a web based server, was used to identify
inhibits nearby nuclear export signal, thus leading to nuclear the SH3 binding domain of apoptin [27]. SH3- Hunter identified
accumulation of apoptin [18,19,20]. Apoptin phosphorylation has the sequences, 81 to 86 (PKPPSK) as the SH3 binding domain.
been proposed to be regulated by Akt-activated CDK-2 and PKC SH3-binding domains have a consensus sequence:(-X-P-p-X-P-)
kinase [21,22,23]. Thus, nuclear localization of apoptin and its with 1 and 4 being aliphatic amino acids, 2 and 5 always-, and 3
interaction with specific signaling proteins plays a crucial role in its sometimes being proline (data not shown) [28].
selective toxicity [19,20,24]. Highly organized recognition of
specific target binding partners by signaling proteins is an Apoptin colocalizes with nuclear phospho-Bcr-Abl and
important aspect of many cellular processes. The specificity of physically interacts with it
these interactions is determined by the physical, structural and We performed immunofluorescent imaging studies to evaluate
chemical properties of the interacting proteins. Therefore, detailed the subcellular localization of apoptin by comparison of the mouse
knowledge about the 3D structures of the involved proteins is myeloid cell line, 32Dp210, stably transfected to express Bcr-
necessary to understand such interactions. Unfortunately, the 3D Ablp210 with the Bcr-Abl non-expressing 32DDSMZ cell line. In
molecular structure of apoptin revealing its precise structure- 32Dp210 cells transfected with GFP-apoptin (Tx, 32Dp210 and
function relationship has not yet been resolved by conventional 32DDSMZ cells transfected with GFP-apoptin), we observed the
crystallography or NMR studies. Determination of the 3D nuclear localization of apoptin detected with anti-GFP concom-
molecular structure requires that the molecule be in crystal form itant to the Bcr-Abl localization detected with Bcr-Ablp210 with
suitable for X-ray crystallographic study or to be of less than Cy3 conjugated secondary antibody (column 2, 3, topmost panel,
30 kD for accurate NMR study. In case of apoptin, the 3D Fig. 2A). Colocalization of nuclear apoptin and phosphorylated
molecular structure could not be attained due to the inability of Bcr-Abl was confirmed in the merged image (column 4, topmost
the molecule to be crystallized, and the nature of the molecule to panel, Fig. 2A). Column 1 in all three panels show DAPI stained
stay in solution as globular multimers [25]. nuclei.
The alternative technique to study protein structures and to Several well-characterized SH3 domains were previously
predict interactions with other proteins is homology modeling identified as potential sites critical to ligand binding [29]. In
and virtual protein docking experiments. This novel method to preliminary experiments, we performed an array-based screening
identify the function of proteins, based directly on the sequence-to- method (TransSignal SH3TM Domain Array1) to identify the
structure-to-function paradigm, is broadly known as computa- interaction of apoptin with the SH3 domains of a known set of
tional protein modeling [26]. While employing this approach, it is proteins (data not shown). This highly stringent SH3 domain
possible to build the best-predicted structure of the protein from its interaction array screening indicated that apoptin strongly

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 Apoptin Structure

Figure 1. Schematic representation of the primary structure and functional domains of apoptin, its cytotoxic potency and
inhibition of Bcr-Abl phosphorylation. (A) The SH3 binding domain is merged within NLS1 (amino acids, aa: 8288). A pictorial representation of
apoptin sequences (UniProtKB/Swiss-Prot entry P54094), LRS = Lecine-Rich Sequence, NLS = Nuclear-Localization Signal, NES = Nuclear Export Signal.
(B) Cytotoxic activity of apoptin on Bcr-Abl positive 32Dp210 cells: 32Dp210 were grown in 96-well plates (104 cells per well). Cells (in triplicates for each
treatment) were treated with 1 mM Tat-apoptin, and Tat-GFP (negative control), or Imatinib for 0, 4, 8, 12, 18 and 24 h periods respectively. The
percentage of viable cells, as assessed by MTT assay indicates that apoptin and Imatinib are both toxic to 32Dp210 cells, and that apoptins cytotoxic
effect favorably compares to that of imatinib. Results are expressed as a percent of cell survival (mean 6 SD). (C) Apoptin inhibits Bcr-Abl
phosphorylation: K562 and 32Dp210 cells were treated with 1 mM Tat-apoptin, Tat-GFP (negative control) or 1 mM imatinib (positive control). Cells
were then harvested after 16 hrs and cell lysates were prepared. Representative Western blots show the expression levels of total and
phosphorylated Bcr-Abl; equal loading was checked by the loading control, eIF4E. The upper panel of bands shows the expression of K562 cells and
the lower panel shows the expression of 32Dp210 cells. Lanes from the left: (1) no-treatment control cell, (2) Tat-GFP treated cell, (3) imatinib treated
cell, and (4) Tat-apoptin treated cell respectively in both cell lines. (D) For quantitation, band intensities from immunoblots were scanned by Image
Quant software (version 5.2, Molecular DynamicsH). During quantitation, the imatinib expression data was omitted in order to enable visualization of
the apoptin effect with greater clarity. Bcr-Abl phosphorylation was significantly inhibited by apoptin. The quantitation data were normalized to the
loading control (eIF4E) and expressed as a ratio of phosphorylated to the total Bcr-Abl and presented as mean 6 SEM of three independent
experiments.
doi:10.1371/journal.pone.0028395.g001

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 Apoptin Structure

Figure 2. Interaction of apoptin with Abl and Bcr-Ablp210. (A) Indirect immunofluorescence showing the nuclear localization of Bcr-Abl.
32DP210 and 32DDSMZ cells were transiently transfected with GFP-apoptin (green) and subjected to (immuno)fluorescence staining and detection.
Apoptin localization was by GFP and Bcr-Abl was detected by staining with Bcr-Ablp210 with Cy3 tagged (red) secondary antibody. Nuclei were co-
stained with DAPI (4, 6-diamidino-2-phenylindole: blue). Column 1 shows DAPI stained nuclei; columns 2 and 3 show the nuclear localization of Bcr-
Abl and apoptin. Column 4 shows the merged image of nuclear co-localized of Bcr-Ablp210 and GFP-apoptin as small clusters (yellow). Abbreviations:
Tx = cells transfected with GFP-apoptin, No Tx = no transfection with GFP-apoptin. (B) To demonstrate apoptin and Bcr-Abl interactions, 510 mg of
GST-apoptin was used in the pull-down assay. The interaction was tested either on 500 mg of total cell lysates from Bcr-Abl expressing 32Dp210 cells,
or on Bcr-Abl non-expressing 32DDMSZ cells. Lanes from the left: (1) the pull-down products of Bcr-Abl in 32DDMSZ extracts (negative control), (2)
32D p210 extract (positive control), (3) 32D p210 extract treated with glutathione-sepharose beads (beads control), and (4) 32Dp210 extract incubated

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 Apoptin Structure

with GST-Apoptin captured with glutathione-sepharose beads. (C) In order to detect apoptin and Bcr-Abl interaction by co-immunoprecipitation
assay, 32Dp210 cells were transiently transfected with GFP-apoptin (3 mg of pEGFP-apoptin plasmid for 26106 cells per transfection using
lipofectamine transfection reagent) and cell lysates were incubated with anti-Bcr-Abl antibody followed by immunoprecipitation by protein G-
sepharose beads; washed IP products were tested for the presence of apoptin (GFP-apoptin: 40 kDa) by immunoblot using anti-apoptin antibody.
Lanes from the left: 1 - GST-Apoptin (positive control), 2 - GFP-apoptin Co-IP from transfected 32Dp210 cells by anti-Bcr-Abl antibody, 3 - Co-IP
supernatant/immunodepleted fraction from transfected 32Dp210 cell lysates, 4 - 32Dp210 transfected with GFP (Co-IP, negative control), and 5 - Co-IP
from 32Dp210 cells without transfection (Co-IP, negative control). (D) The Abl SH3 domain in Bcr-Ablp210 facilitates Bcr-Abl interaction with apoptin.
32DDSMZ cells were transfected with various Bcr-Abl mutant constructs by lipofectamine using 34 mg purified plasmid DNA per 26106 cells. Specific
mutant clones of transfected cells were selected by G418. Pull-down assays were performed 710 days following the selection and expressed proteins
were detected by immunoblotting. The upper representative immunoblot shows various mutants of Bcr-Abl expressed in various 32DDSMZ clones.
The lower immunoblot shows results of GST-apoptin pull-down assay. The Src-homology domain mutant of Bcr-Ablp210 and GST-apoptin were used
in this pull-down (.,) experiments using lysates from various Bcr-Abl mutant protein expressing 32DDSMZ clones. The protein-protein complexes
were analyzed for apoptin interaction by immunoblotting with rabbit anti-Bcr antibody. As seen (lane 69), the presence of an intact SH3 domain in
the Bcr-Abl molecule is essential for its interaction with apoptin. Some degree of Bcr-Abl.,apoptin interaction was also seen in the Abl-SH3 domain
in Bcr-Abl, which was partially modified by single aa substitution (lane 10).
doi:10.1371/journal.pone.0028395.g002

interacted with the SH3 domain of Abl. This preliminary deleted SH3, single amino acid (aa) substitution at SH2 and intact
observation was further substantiated by pull-down assay and SH1, and (iv) Bcr-Ablp210P1013L-R1053L: had single aa substitu-
co-immunoprecipitation (Co-IP) studies using the Bcr-Ablp210 tion at the SH2 and SH3 domains respectively and intact SH1
stably expressing 32Dp210 cells and compared to the Bcr-Abl non- domain. Mutants were selected according to their specific nature of
expressing 32DDSMZ cells (Fig. 2B). For the GST-pull down assay SH-domain mutations to probe if apoptin interacted with the SH3
recombinant GST and GST-conjugated apoptin were purified domain of Bcr-Abl. Full length Bcr-Abl, and its derivates with intact
from IPTG stimulated transformed bacterial clones harboring the SH3-domain interacted with GST-Apoptin and were pulled-down
respective plasmids. The membrane was probed with anti-Bcr-Abl by glutathione sepharose beads, while other mutants lacking intact
primary antibody. A representative blot from such an experiment SH3 domain failed to show such interaction.
shows the presence of Bcr-Abl (Lane 5) in the GST-Apoptin pull-
down product (220 kD, Fig. 2B). This 220 kD protein pulled Bioinformatics analysis of molecules known to interact
down by apoptin, was confirmed by the presence of a similar band with (Bcr-)Abl
in the lysates of 32Dp210 cells with stable expression of Bcr-Ablp210.
Global gene expression data for K562 cells was analyzed as
This in vitro assay demonstrated apoptin interactions with Bcr-Abl.
published [31]. Pathway analysis and visualization was performed
Non-specific interactions in the absence of GST-Apoptin (beads
using the GenMapp and pathVisio bioinformatics tool [32,33].
control, lane 4) were not detected. We further confirmed Bcr-Abl
The BioCarta pathway for the Bcr-Abl regulated genes in K562
and apoptin interactions by Co-IP when GFP-Apoptin was
cells was analyzed and visualized using PathViSio (Fig. 3A).
transiently expressed in Bcr-Abl expressing 32Dp210 cells (Fig. 2C).
Another bioinformatics tool, Ingenuity Pathways Analysis was
used to build and to identify the directly- or indirect interacting
Apoptin interacts with Bcr-Abl via a specific motif network of molecules (Fig. 3B) [34]. Bioinformatics analyses were
To identify the precise nature of apoptin and Bcr-Abl interaction validated experimentally for some of the molecules present in the
in CML cells, we mapped the sites on apoptin responsible for pathways and in the networks.
interaction with specific region of Bcr-Ablp210. The murine bone
marrow derived 32DDMSZ, 32Dp210 cells and the human CML cell
line K562 were grown in appropriate media and transfected with Apoptin down-regulates the Bcr-Abl kinase activity and
different apoptin mutant constructs (Materials and Methods, modulates the phosphorylation of downstream kinases
[18,19]). The expression of these mutant derivatives of apoptin To study the down-stream effects of apoptin and Bcr-Abl
tagged with an N-terminal GFP was verified by SDS-PAGE and interaction, we examined the expression and phosphorylation
immunoblotting with mouse monoclonal anti-apoptin antibody. status of Bcr-Ablp210 and other major down-stream Bcr-Abl targets
Apoptin was immunoprecipitated by murine anti-GFP antibody like STAT5, CrkL, c-Myc and Akt in mouse (32Dp210) and human
from lysates of transfected cells expressing various mutations of (K562) CML cell lines. In immunoblotting experiments, we
apoptin with murine anti-GFP monoclonal antibody and the measured the phosphorylated and total proteins. The overall
protein complexes were analyzed to detect the presence of Bcr- results indicate that apoptin induced inhibition of Bcr-Abl
Ablp210 by using rabbit monoclonal anti-Bcr antibody. Bcr-Ablp210 (auto)phosphorylation (Fig. 1C), down-regulated STAT5
was found in the immunoprecipitates of full-length apoptin and (Fig. 3CD) and CrkL (Fig. 3GH) and activated Akt (Fig. 3EF).
apoptin derivatives that harbored amino acids from 74100 These results are consistent with global gene expression pattern
(including the Proline-rich sequence: PRS), implying that this observed in untreated K562 cells (Fig. 3AB). In all experiments, a
region of apoptin is important for interaction with Bcr-Ablp210 wt comparison was done with imatinib treated cells (positive control),
(data not shown). Interestingly, in this model system, the mutants a known Bcr-Abl inhibitor and clinically-used CML-therapeutic.
Ala-108 and Glu-108 have a Thr-108 residue of apoptin We quantified the relative phophorylation level of Bcr-Ablp210 by
replacement by alanine or glutamine respectively; these replace- immunoblotting with phospho-Bcr-Abl-specific antibodies and
ments render apoptin as non-phosphorylatable and are claimed by thus we could assess the inhibition of activated Bcr-Abl by apoptin,
some authors to be non-toxic to cells [30]. Subsequently, the specific which was highly significant (p,0.01, 0.04) in both cell lines.
interactions between full-length GST-conjugated apoptin and Bcr- STAT kinases serve a dual role of signal transducers and
Ablp210 or various SH-domain mutant-constructs of Bcr-Abl activators of transcription. Among the large family of over 30
expressed in 32DDMSZ cells were studied (Fig. 2D). The mutants STAT proteins, STAT5 has been identified as a key factor
included: (i ) Bcr-Ablp210DSH2: had an intact SH3, deleted SH2 and involved in anti-apoptotic signaling and malignant transformation
intact SH1, (ii ) Bcr-Ablp210DSH2 DSH3: had a deleted SH2, in CML. Here we show that STAT5 phosphorylation was
deleted SH3 and intact SH1, (iii) Bcr-Ablp210DSH3-R1053L: had a markedly reduced in K562 cells following Tat-apoptin treatment,

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 Apoptin Structure

Figure 3. Visualization of pathways, interacting network, and validation of selected downstream regulators. (A) Bcr-Abl and its
downstream effectors are shown by BioCarta pathways. Global gene expression data of K562 leukemia cells was taken from public database,
analyzed, and visualized (GenMAPP). The expression values of signal log base e ratio (SLR) are shown outside the colored boxes. The software
generated color codes denote up-regulation (dark-red) and down-regulation (blue-green). (B) Direct and indirect interacting network associated with
Bcr-Abl was built utilizing global gene expression data and visualized by IPA. The up-regulated genes are shown in red and down-regulated genes are
shown as green. The gene expression values (SLR) are also shown. (C, E, G) Apoptin induced inhibition of Bcr-Abl phosphorylation leads to the down-
regulation of downstream regulators, STAT5, Akt, and CrkL respectively. K562 and 32Dp210 cells were treated with 1 mM Tat-apoptin, Tat-GFP
(negative control) and 1 mM imatinib (positive control) and cell lysates were prepared by harvesting cells after 16 h. Representative Western blots
(divided into upper and lower panels representing the K562 cells and the second panel represents the 32Dp210 cells) show the ratio of the expression
levels of phosphorylated and total STAT5 (D), Akt (F), and CrkL (H) respectively. In all the immunoblots, lane 1 from no-treatment control cells, lane 2
is from Tat-GFP treated cells, lane 3 is from Tat-apoptin treated cells respectively for both cell lines. STAT5 phosphorylation was significantly inhibited
by apoptin indicating that apoptin induced inhibition of Bcr-Abl phosphorylation decreases the activation of STAT5 through phosphorylation. On the
other hand, Akt phosphorylation was higher, indicating that apoptin induced Akt activation, as previously published. For CrkL, apoptin induced
inhibition of Bcr-Abl phosphorylation lead to the down-regulation of CrkL resulting in lower phosphorytion indicating that apoptin decreases the
activation of CrkL, a down-stream substrate of Bcr-Abl. For quantitation, band intensities were scanned by Image Quant software (version 5.2,
Molecular DynamicsH). During quantitation, imatinib expression data was omitted in order to enable greater visualization of the apoptin effect. The
quantitation data were normalized to the loading control (eIF4E/b-tubulin) and expressed as a ratio of phosphorylated to the total protein and
presented as mean 6 SEM of three independent experiments.
doi:10.1371/journal.pone.0028395.g003

which was comparable to imatinib (Fig. 3CD). Similar statistically (Fig. 3GH). For analysis of the pro-apoptotic effect of apoptin in
significant results (p,0.03) were obtained in Bcr-Ablp210 express- Bcr-Abl expressing cells, we examined its effect on the signaling
ing mouse cell line 32Dp210 (Fig. 3CD). Moreover, we also studied protein Akt and compared it to imatinib (Fig. 3EF). Interestingly,
the downstream consequences of Bcr-Abl inhibition by apoptin on although Akt is known mediator of cell survival, we observed a
the phosphorylation status of CrkL. This 39 kD protein is involved marked augmentation of Akt phosphorylation 16 h after either
in b-integrin signaling and is a prominent substrate for activated apoptin or imatinib treatment of K562 and 32Dp210 cells
Bcr-Abl kinase [35]. We observed a significant (p,0.04) inhibition (Fig. 3EF). It is possible that the activated Akt may still act in an
of CrkL phosphorylation in K562 cells treated with 1 mM Tat- anti-apoptotic manner in Bcr-Abl expressing cells if re-located to
apoptin for 16 hrs comparable to imatinib treated cells (quanti- the nucleus as previously proposed [23,36,37]. Our results were
fication not shown for imatinib) (Fig. 3GH). Marked inhibition of consistent with the global gene expression data for STAT5, CrkL,
CrkL phosphorylation was also in Bcr-Ablp210 expressing 32Dp210 and Akt (Fig. 3AB).

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 Apoptin Structure

Table 1. List of template proteins used to build apoptin model.

PDB codes* PROTEINS

1WLS_A L-asparaginase I homologue from Archacea (pyrococcus horikoshii)

1OQY_A Human UV excision repair protein RAD23 homolog A
1Q9J_A Ml2640c from mycobacterium leprae
Apoptin PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis.
1E3I_A Murine alcohol dehydrogenase, class II
1E3L_A P47H mutant murine alcohol dehydrogenase, class II
2GYZ_A Neurotrophic factor artemin, isoform 3
2GYR_A Neurotrophic factor artemin, isoform 3
2GH0_C DNA-directed RNA polymerase alpha chain
1QZE_A UV excision repair protein RAD23 homolog A
1WNF_A PH0066 (L-asparaginase) from Archacea
2ASK_A Human artemin
2UYQ_A A viral protein encoded by the VP3 gene of Chicken Anemia Virus

*Data source: Protein Data Bank (RCSB-PDB).

doi:10.1371/journal.pone.0028395.t001

Development of homology based three-dimensional main chain atoms. This model of apoptin (aa:1121), most
model of apoptin residues, about 81.8% of the residues (81 residues), were in the
In this part of the study, unknown 3D structure of apoptin was most favored regions, 16.2% in the additional allowed regions,
approximated by a comparative- or homology protein modeling. 2.0% in the generously allowed regions, and no residues were fall
To this end, we used the protein sequence (target) based on the in the disallowed regions according to Ramachandran plot
known 3D structure of proteins with domains that have related (Fig. 4D). According to Procheck, the overall G-factor was about
peptide sequences (Table 1). The 3D structures of several known 20.35. After examining the accuracy of the model, all atoms of the
templates (Table 1) with identified partial homology to apoptin molecule were locked, hydrogen atoms were added and molecular
were used to build the 3D structure. To build the apoptin model, mechanics (MM2) and molecular dynamic simulations was
we used several modeling programs such as Modeller [38,39] and performed at 1000 K for 50 ps simulation duration with 0.001
DeepView [40], as well as project mode and the alignment mode simulation time-step (ps). All theoretical calculations and visual-
of Swiss Model web based server [40]. After numerous trials using ization were performed using Scigress Explorer Ultra associated
different templates, we were able to build the full-length apoptin with the Gaussian03 software [42,45,46].
model. It is worth mentioning that model quality was different This model was used to examine solvent accessible surface area
when we used different chain of the same structure as a template. (Fig. 4E) to identify the surface (large patches, cream color, of
To understand this difference in model quality, we superimposed hydrophobic areas) of the protein that are involved in interactions
two chains in one of the templates (for example: PDB code: 1WLS, with other proteins and hydrophilic regions that involved in
chains A and B) and noticed the differences between two chains as hydrogen bonding, hydrogen bond acceptors (red color) and
jugged by the RMS value (0.877 A) when two chains were hydrogen bond donors (blue color). This model was further used to
superimposed. The difference in the RMS value may be due to the perform virtual docking experiments to examine and to under-
missing residues in some cases and/or due to differences in stand the interactions between apoptin and Bcr-Abl.
resolution between two chains. The Swiss Server Alignment mode
provided better results when multi-sequences were used. The T- Virtual docking of Bcr-Abl and apoptin model
Coffee or ClustalW2 multiple sequence alignment tools [41] were To examine protein-protein interaction between apoptin model
used to align a group of five or six sequences from a group of and the 3D structure (PDB code: 2ABL) of Bcr-Abl, molecular
templates (Table 1). Modeller [38,39] provided the best results. docking experiments were performed using ClusPro [45,47] and
One of the best models was used for further studies (Fig. 4BC). Hex [48] web based protein docking servers. The ClusPro
The coordinates of this model are submitted as supporting provided about ten structures. One of the lowest energy structures
materials (Coordinates S1, S2, S3). (Fig. 5AB) was used for further analysis. All atoms are locked and
Subsequently, a Ramachandran plot was performed to verify hydrogen atoms were added and energy optimization was
the quality of the model (Fig. 4D) [40,42,43,44]. The N-Ca and performed. Finally, interacting residues between two molecules
Ca-C bonds in a polypeptide chain are relatively free to rotate. that are within 2.5 A of each other were identified and given in the
These rotations are represented in the plot by the torsion angles Table 2 and in Table 3 corresponding hydrogen bond distances
phi (w) and psi (y), respectively. The structure was examined for are presented.
close contacts between atoms for each of these conformations.
Atoms were treated as hard spheres with dimensions correspond- Shape and sequence similarity of apoptin and the SH2
ing to their van der Waals radii. Therefore, angles that cause domain of CrkL
spheres to collide correspond to sterically disallowed conforma- CrkL domains were identified using Prosite [49], a web based
tions of the polypeptide backbone. Disallowed regions involve server. Prosite identified SH2 (aa 14102) and SH3 (aa: 123183)
steric hindrance between the side chain methylene group and domains of CrkL. Sequence alignment of apoptin and the SH2

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 Apoptin Structure

Figure 4. Sequence alignment and 3D model of Apoptin (aa: 1121). (A) A representative sequence alignment between apoptin residues
and the residues of one of the templates from a group of templates (Table1) is shown. (B) Solid ribbon view of full-length (aa: 1121) of 3D model for
apoptin and its amino and carboxyl terminals are shown. (C) Space filling view of apoptin model, showing the potential hydrophobic proline rich
interacting area (PKPPSK, aa: 8186, pink colored region, top right) is shown. (D) Ramachandran plot showing the N-Ca and Ca-C bonds in the
apoptin polypeptide chain represented by the torsion angles phi (w) and psi (y); quality of the model was examined by this plot (all atoms are within
the allowed regions) and by the G-factors values (the overall value for G-factors is 20.35). (E) Solvent accessible surface area shows the regions of
hydrophobic (large cream colored region at the surface) where protein-protein interactions could occur and the hydrophilic regions that are involved
in hydrogen bonding, hydrogen bond acceptors (red color) and hydrogen bond donors (blue color). Additional information on apoptin structure
could be found in Coordinates S1, S2, S3.
doi:10.1371/journal.pone.0028395.g004

domain of CrkL were performed. Interestingly, we observed that virtual docking experiments between the structure of SH2 domain
the sequence of apoptin was somewhat similar (identical residues of CrkL and the structure of Bcr-Abl (PDB code: 2ABL).
21.7%, and similarity 40.6%) to that of SH2 domain of CrkL, and
apoptins proline-rich segment (aa: 8188) was found to be within Discussion
this aligned region of SH2 domain. We then compared the shape
of known 3D structure (PDB code: 2EO3) of SH2 domain of CrkL The 3D structure of apoptin has been unknown due to
and apoptin model. Sequence alignment structural similarities are numerous reasons (lack of a suitable crystals, multimerisation in
shown in figure 6A, B, and C respectively. We also performed the solution), furthermore apoptin modeling is challenging due to the

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 Apoptin Structure

Figure 5. Modeled interactions between apoptin and Bcr-Abl. (A) Shows the interaction between apoptin and the SH3-domain of Bcr-Abl
(solid ribbon view, showing the two terminals of two proteins) obtained by performing virtual docking experiment between apoptin model and the
X-ray structure of Bcr-Abl-SH3 domain (PDB: 2ABL). (B) Shows the space filling docking view of the interactions between apoptin (pink) and the SH3-
domain of Bcr-Abl (blue), the 13 residues (red) of Bcr-Abl and 13 residues (light blue) of apoptin that are within 2.5 A to each other; some of the
proline-rich (PxxP) SH3-binding residues (Table 2) are present and at least five direct hydrogen bonding are possible in between them (Table 3).
Additional information on apoptin interaction with BcrAbl could be found in Coordinates S4, S5, S6.
doi:10.1371/journal.pone.0028395.g005

low number of suitable templates. We have been able to build a X-ray crystal structure of Bcr-Abl (PDB code: 2ABL). First,
model of apoptin by applying a comparative or homology protein accessible surface area for apoptin was identified. As shown in
modeling approach despite low identity (about 31%) and similarity figure 4E, the large cream colored area is the hydrophobic region,
(about 52%) of the templates. Figure 4AC, and E shows the the sites for protein-interaction, purple-red areas and blue areas
sequence alignment of the templates, ribbon view, space filling full- are hydrophilic regions, purple-red indicates hydrogen bonding
length model of apoptin, and Ramachandran plot, and solvent acceptors (for example, C = O) and blue regions indicate hydrogen
accessible surface area respectively. This model was used to bond donors (for example, N-H or O-H).
virtually examine various binding interactions with Bcr-Abl by Using this model, we have been able to identify the nature of
performing virtual docking experiment between apoptin and the interactions and hydrogen bonding between the residues of SH3
domain of Bcr-Abl and apoptin (Table 3). Subsequently, we have
experimentally verified the observed interaction between apoptin
Table 2. Interacting amino acid residues of apoptin and Bcr- and the SH3 domain of Bcr-Abl oncoprotein. In this model
Abl. system, 13 aa (Fig. 5B, red) of SH3 domain of Bcr-Abl are
approximated within 2.5 A of apoptin and 13 aa (Fig. 5B, light-
green) of apoptin are within 2.5 A of Bcr-Abl residues.
Apoptin Interacting residue BCR-ABL residue Interestingly, some of the proline rich PxxP sequences (aa: 81
86, QPKPPSKKR) (Fig. 5AB) are involved in these interaction
Thr8 Lys29
among other nearby residues and at least five pairs of direct
Lys82* Glu68
hydrogen bonding are possible between them (Table 2). This low-
Pro83* Ser66, Glu68
Lys86* Pro63
Table 3. Amino acid residues forming hydrogen bond
Lys87* THR62
between apoptin and Bcr-Abl.
Thr108 Ser71
Arg111 His74
Pro112 His74 Apoptin residue BCR-ABL Hydrogen Bond distance (A)

Thr114 Tyr84, Leu85 Thr8 Lys29 1.983

Ala115 Val77 LYS82* Glu68 1.86
Lys116 His74 Lys86* Glu98 2.072
Arg118 Ser 78, Glu100 Lys116 His74 1.912
Ile119 Pro76 Arg118 Glu100 2.102

*The SH3 interacting amino acids in the proline rich PxxP region of apoptin are *Hydrogen bonding forming residues between the Bcr-Abl and the proline rich
marked as bold. PxxP region of apoptin are shown in bold.
doi:10.1371/journal.pone.0028395.t002 doi:10.1371/journal.pone.0028395.t003

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 Apoptin Structure

Figure 6. Sequence alignment and interactions between apoptin and adopter proteins CrkL, Akt1 and STAT5. Space filling views and
similarity in sequences between apoptin and the SH2-domain of the adopter protein CrkL (A), apoptins SH3-binding domain residues (B), 81 to 86
(PKPPSK), are within the SH2-domain of CrkL (C). In addition, the similarity in sequences between apoptin and the adopter proteins Akt1 (D) and
STAT5 (E) suggesting that apoptin might directly interact in the CrkL, Akt1 and STAT5 interacting sites in addition to the SH3-binding domain of Bcr-
Abl and could block further propagation of survival and proliferation signaling.
doi:10.1371/journal.pone.0028395.g006

resolution model provides information about the interaction proliferating human and murine cell lines have a high cytoplasmic
between Bcr-Abl and apoptin and it helps us to explain the Bcr-Ablp210 pool and thus the cell culture condition mimic the
probable mode of action; moreover, our experimental pull-down blast crisis stage of CML. Furthermore, as in CML, the central
assay and co-immunoprecipitation studies confirm occurrence of mitogenic Ras-MAPK cascade is also activated, similarly as in our
those interactions in cell nuclei. model cell lines. Our findings corroborate well with previous
We not only show for the first time, that Tat-apoptin, a cell- studies, by Kardinal and colleagues, involving a similar approach
penetrating conjugate of apoptin strongly binds to the SH3 directed towards the Grb2-SoS-Ras-MAP kinase (Erk) pathway
domain of Bcr-Abl, but also it modifies the phosphorylation status [50]. In these experiments, small, high affinity peptides blocking
and thus the activity of Bcr-Abl, and several of its downstream the N-terminal SH3 domain of Grb2 were applied. Their results
targets. These changes lead to the anti-proliferative effect and indicate that peptide based inhibitor of Bcr-Abl kinase or its down-
induction of intrinsic apoptotic pathways in rapidly dividing CML stream targets could be valuable anti-CML tool if combined with
cells. Using human CML cell line, K562 and Bcr-Ablp210 conventional cytotoxic therapy [50]. We have also observed that
expressing murine cell line 32Dp210 as models, we observed that apoptin-derived peptides capable of interaction with SH3 domain
these cells are significantly responsive to apoptin. These highly are toxic against Bcr-Abl expressing cells (data not shown). We

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 Apoptin Structure

have further demonstrated that apoptin, unlike Imatinib/Gleevec, STATs act as regulators of cell proliferation [63]. The N-
was effective both against Bcr-Abl positive and also Bcl-Abl terminal regulatory region of Abl protein contains the SH2 and
negative cells. We thus hypothesize that apoptin-based therapeu- SH3 domains which are important for the regulation of activity in
tics would be not only more effective, but have the additional vivo [64]. We have previously reported that apoptin productively
advantage that they would be less prone to the development of interacts with the SH3 domain of p85 regulatory subunit of PI3-K
resistance. [18]. In the current study, we report for the first time that apoptin
Activation of Bcr-Abl is critical for the development of CML. inhibits STAT5 activation in Bcr-Abl expressing cell lines.
Different downstream molecules and pathways such as the Grb2- Furthermore, since STAT5 also regulates the Bfl-1 family gene
Ras-Raf-Mek1/2 Erk pathway, the PI3 kinase pathway involving A1 that reportedly collaborates with c-myc and is required for Bcr-
Gab2 [51,52,53,54,55], the Jak2-STAT3 pathway [56,57], and Abl transformation [65], this observation strongly supports the role
the Bcr-Abl-STAT5 pathway [58,59] are implicated as shown in of apoptin as a proliferation inhibitor of CML cells.
figure 3A. Using bioinformatics approaches, we visualize the Different components of the Bcr-Abl downstream pathways are
relationship between these component molecules and pathways involved in the pathogenesis of CML and are highly active
using global gene expression data. A comprehensive analysis of compared to normal cells. Hyperactivation of STATs, Ras-MAPK
molecular interactions of Bcr-Abl target molecules that are either or CrkL-integrin pathways lead to the development of character-
directly (solid lines or solid lines with arrows) or indirectly (broken istic CML pathologic features. Apoptin affects many of these
lines or broken lines with arrows) involved are shown in an signaling events, and thus it is well suited for targeting the cellular
interaction network (Fig. 3B). Interrelationship between molecules signaling environment of Bcr-Abl expressing cancer cells. In
is clearly visualized their activated (pink color) or repressive (green addition, apoptin is a good candidate to serve as a model/lead
color) states. In this figure, expression values are also shown. As molecule for the development of smaller peptides or peptidomi-
shown in diagram 3A, the network of genes and proteins is very metics that would target multiple cell proliferation and anti-
complex and in the context of drug design it is essential to consider apoptotic pathways. This may be of advantage also for CML-
these interrelationships to avoid drug toxicity. treatment because advanced highly mutated CML-cells may no
In our previous studies, we have shown that direct apoptin-Akt longer solely rely on Bcr-Abl as the driver of cell proliferation
interaction initiates nuclear trafficking of Akt. Interestingly, (hence, acquired resistance to Imatinib). Apoptin acts on multiple
nuclear Akt, instead of activating an anti-apoptotic response targets related to cell proliferation by blocking their association
initiates apoptosis by a process that is only partially understood with Bcr-Abl rather than binding. Thus, this is an alternative
[36]. Our observation corroborates well with recent data from approach to the conventional target based rational drug design to
other studies, showing that Akt inhibitors have been only block the interactions of adapter molecules rather than binding a
moderately successful in experimental cancer therapy [60]. small molecule to the active site. When a small molecule diffuses
Furthermore, similar to the earlier observed nuclear transfer of into a macromolecule, it alters the shape and size of the
Akt, we have also observed the nuclear transport of apoptin- macromolecule leading to its conformation change. These changes
interacting protein Bcr-Abl. We hypothesize that nuclear re- in shape, size, and dynamics could lead to the activation/
location of Bcr-Abl may markedly affect its biologic properties. deactivation of undesired bio-molecules that in turn may yield
The adaptor proteins CrkL forms a complex with Bcr-Abl detrimental effects.
leading to Bcr-Abldependent phosphorylation of CrkL and To improve the potency and specificity of small apoptin-like
subsequent phosphorylation of c-Cbl may contribute to Bcr-Abl peptides, designing new small molecules with proper shape,
dependent activation of PI3 kinase [61]. Multiple downstream number of proline residues in appropriate positions, and capability
targets of PI3 kinase have been identified. But apoptin interaction of nuclear trafficking is essential. To avoid undesirable drug
might block complex formation between the adapter protein CrkL effects, repetitive evaluation of potency, examination of global
and Bcr-Abl. Nuclear localization of apoptin is essential to block gene expression, and extensive bioinformatics analysis are
the complex formation between Bcr-Abl and CrkL. We have indispensable until a drug-like molecule with desired properties
examined the nuclear localization of apoptin as shown Figure 2A. is achieved. Present study, model-structure-function relationship,
As previously mentioned, apoptin is a cytoplasmic molecule but provides such opportunities to design next generation of apoptin-
nuclear localization occurs upon phosphorylation at Thr108 in like molecules with desired properties. We are aware of limitations
transformed cells, thus in CML-cells it is predominantly nuclear. of computational modeling of protein structure. However since we
These interactions and trans-activation of CrkL and c-Crk II by are able to confirm by biochemical methods the predicted
activated Bcr-Abl kinase and their functional consequences are intermolecular interactions, we are convinced that the provided
well documented [35,62]. In the current study, we demonstrated model is highly accurate.
for the first time that apoptin inhibits phosphorylation of CrkL in
Bcr-Abl expressing cells. This observation indicates that apoptin Materials and Methods
can indirectly affect growth-supportive role of phosphorylated
CrkL by inhibiting Bcr-Abl kinase. Overall, these observations Three-dimensional/3D modeling
signify apoptin as a negative-modulator of Bcr-Abl kinase activity, The homology modeling approach was used to generate 3D
and indirectly, of the multiple cell proliferation and anti-apoptotic structures of apoptin, a viral protein encoded by the VP3 gene of
pathways that are fuelled by Bcr-Abl. We also consistently Chicken Anemia Virus that is composed of 121 amino acids
observed the activation of Akt upon apoptin and/or imatinib (13.6 kDa). The crystal structure coordinates of the PDB id 1WLS,
treatment in Bcr-Abl expressing cells. Akt is a downstream target L-asparaginase from the hyper-thermophilic archaeon Pyrococcus
for Bcr-Abl kinase and known to interact with apoptin. However it horikoshii was used as one of the templates. The sequence of apoptin
also functions independently of Bcr-Abl, for example, it is one of has about 31% identity and about 52% similarity with the
the key effectors of the PI3-K/PDK1-2 pathway upon cell sequence of the PDB id 1WLS. As mentioned earlier, different
membrane triggering. Beside its pro-survival function, activated approaches were used to build the apoptin model. For alignment
Akt if located in the nucleus, will promote cell death rather than mode (Swiss Model web based server), five sequences, including
cell survival [23,36,37]. apoptin, with known 3D structures were aligned using T-Coffee

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 Apoptin Structure

Multiple Sequence Alignment Tool [41] and then submitted for Pathway, Interacting Network using and Global Gene
model building. For project mode (Swiss Model web based server), Expression data
the DeepView Tool [40] was used to align sequences of known Pathways and gene/protein interacting networks were exam-
structure, then apoptin sequence was threaded to the crystal ined from a global prospective using bioinformatics tools and
structure of PDB id 1WLS and then submitter for model building. microarray gene expression data, such as GenMapp and
Modeller [38,39] a web-based server was also used in model Ingenuity Pathway Analysis. We used the publicly available gene
building. Several other computer programs [42,43,44,66] were expression data from K562, a leukemia cell line expressing Bcr-
used to build and process the apoptin model using 121 amino acids Abl. Two input files specific for each bioinformatics tool were
sequence. Several models building were performed using different prepared. Expression of some of important molecules was
templates and accuracy was examined. One of the best models was validated experimentally in the presence and absence of apoptin.
used for further studies. All other calculations including molecular
dynamic simulation and visualization of 3D structure were
performed using Scigress Explorer Ultra [46]. After building the Cell lines, plasmids, Cell death and cell proliferation
3D model of apoptin, all atomic positions are locked and required assays, antibodies and reagents
hydrogen atoms were added to the backbone structure of the All cell culture media and supplements were from Gibco BRL.
apoptin molecule and performed molecular mechanics calcula- 32DDSMZ and 32Dp210wt:b3:a2/e13:a (denoted as 32Dp210) [67]
tions and then performed molecular dynamics simulation at Bcr-Abl variant were grown in RPMI-1640 cell culture medium
1000 k for 50 ps for further optimization. One of the best apoptin 500 ml supplemented with 20% FBS (Hyclone), 0.05 g penicillin/
models was used to examine the solvent accessible surface area. A streptomycin, 10 mM HEPES (2-(4-hydroxyethyle)-1-piperaziny-
docking file with pdb extension of apoptin molecule was prepared l)ethanolsulfonsaure), 2 mM L-Glutamine, 0.13 mM L-Asparagin,
without hydrogen atom to perform molecular docking p17exper- 0.05 nM 2-Mercaptoethanol, 1 mM Na-Pyruvate, and 3 ml 1006
iments to examine the interaction between apoptin model and the non-essential amino acids. 32DDSMZ cells are strictly dependent on
Bcr-Abl oncoprotein using 3D structure of the protein (PDB code: murine interleukin 3, so the media was supplemented with 10%
2ABL). supernatant from WEHI-3B cells [67]. The human CML cell line
K562 (ATCCH # CCL-243TM) was cultured in ATCC recom-
Validation of 3D Model mended Iscoves modified Dulbeccos medium with 4 mM L-
After building the 3D apoptin model, the Protein Structure & glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 10%
Model Assessment Tools [42,43,44] was used to verify the quality FCS (v/v) and antibiotics [68,69]. All cells were grown at 37uC
of the apoptin model. This tools is capable of verifying a number with 5% CO2 in a humidified incubator and maintained in a
of aspects of model qualities such as (1) Local Model Quality logarithmic growth. The following plasmids were used: GFP, GFP-
Estimation (anolea atomic mean force potential, empirical force Apoptin (apoptin cloned into pEGFP-C1 vector, Clontech), GST,
field, composite scoring function for model quality estimation); (2) GST-Apoptin (apoptin cloned into PGEX-2T vector, Amersham
Global Model Quality Estimation (all-atom distance-dependent Biosciences), apoptin mutants were previously described [19]. The
statistical potential); (3) Stereochemistry Check (protein structure p210Bcr-Abl mutants (kind gift from Dr. T. Skorski, Temple
verification, stereochemical quality check; min. resolution 2.5 A) University, USA), DSH2 (deletion of the SH2 domain),
and (4) Structural Features (secondary structure, geometrical DSH3+DSH2 (deletion of both the SH3 and the SH2 domains),
features, and solvent exposure assignment, analysis of protein DSH3+R1053L (deletion of the SH3 domain and single aa
structure motifs). Based on these assessment results, model quality substitution in the SH2 domain) and P1013L+R1053L (single aa
was evaluated according to the Ramachandran plot and the amino substitutions in the SH3 and SH2 domains), cloned in
acid residues in the allowed, disallowed region and overall G- pSRaMSVtkneo vector as described [70]. Plasmids were propa-
factor. gated either in BL21(DE3)pLysS or DH5a E. Coli strains, upon
transformation by CaCl2 chemical method. Plasmid isolation
Molecular Docking of Apoptin model to the 3D Structure (positive clones) was done using Qiagen Maxi-prep kits. Cell
of SH3 domain of Bcr-Abl proliferation was assessed by MTT assay, and cell death was
Three (Hex, ClusPro, and AutoDock) computer programs measured by propidium iodide uptake as previously described
[45,48] were used to perform the docking experiments. All [71].
docking programs required two separate input files for the two Chemicals and antibodies were purchased from Sigma-AldrichH
molecules, apoptin model and 3D structure of SH3 domain of Bcr- Inc. (Sigma, Oakville, ON, Canada) AbcamH Inc. (Cambridge,
Abl, without hydrogen atoms with pdb extension. The ClusPro MA, USA) or Cell Signaling TechnologyH, Inc. (Danvers, MA,
provides greater and accurate information. Several apoptin models USA). The following antibodies were used: murine/rabbit anti-
were used to perform docking experiments using ClusPro docking Bcr-Abl/anti-Bcr (monoclonal: AbcamH Inc.), murine anti-Akt
server. After the docking experiments, the ClusPro server provided (monoclonal) and the murine monoclonal anti-apoptin antibody
about 10 structures. The lowest energy docking structure was used (kind gift from Dr. D. Jans, Australia).
for further studies. After the docking experiments, all other
modeling and calculations were performed using DeepView and Transfection of mammalian cells
Scigress Explorer Ultra. All atomic positions of the lowest energy Different mammalian primary and cancer cell lines were
docking structures are locked and required hydrogen atoms were transfected with the desired plasmids by LipofectamineTM2000
added to the structures and performed molecular mechanics (InvirogenH Canada Inc. Burlington, Ontario, L7P 1A1) reagent.
calculations to minimize the energy for the added hydrogen atoms. The cells were plated in an antibiotic free medium 24 h prior to
This minimize structure was used to examine and to identify the transfection and plasmid DNA was added to 100 ml of medium in
interacting residues between apoptin and the Bcr-Abl molecules. a tube at the recommended concentrations (typically 2 mg for a 12
Hydrogen bonding of the interacting residues between the two well plate and 5 mg for a 6 well plate) at the time of transfection.
proteins was also examined. These molecular interactions were The Lipofectamine reagent (5 ml for 12 well and 10 ml for 6-well
further verified biochemically. plate) was diluted in 100 ml of medium in a second tube. Next, the

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 Apoptin Structure

DNA and Lipofectamine reagents were mixed after 5 min and Bcr-Abl multiplex kinase assays
then incubated for 20 min at room temperature to allow the The phosphorylation status of Bcr-Abl, STAT5, CrkL and Akt
formation of DNA liposome complexes. Following the incubation, were measured by scanning the signal strength of phosphorylated
the DNA-lipid mixture was gently added directly to the cells that proteins on Western blot membranes. Kinase reaction was
had previously been rinsed with PBS and replaced with fresh performed in K562 and 32Dp210 cells by overnight (16 h)
medium. incubation with Tat-apoptin (test), ImatinibH (positive control)
Tat-GFP, or no treatment (negative controls) and cell extracts
GST-pull down assay and protein identification, co- were resolved by SDS-PAGE (10%) and detected by immuno-
immunoprecipitation (Co-IP), Western blotting blotting using respective phospho-specific antibodies or antibodies
The GST and the recombinant GST-apoptin proteins were detecting both phosphorylated and non-phosphorylated kinase. A
purified according to the manufacturers protocol using glutathi- high-resolution scanner (STORM 860: Molecular DynamicsH:
one sepharose beads (Amersham BiosciencesH). GST-pull down Amersham Pharmacia Biotech) was used to scan the immunoblot
assay was performed to detect the interacting partners of apoptin. membranes after ECL detection and individual band intensities
Briefly, either purified GST or GST-apoptin along with total were measured by Image Quant (Version 5.2) software (Molecular
32Dp210 or K562 cell lysate (by sonification) were immobilized on DynamicsH). Band intensities were normalized to the respective
loading controls and expressed as a ratio of measured values for
glutathione sepharose beads overnight at 4uC in IP buffer with
phosphorylated proteins versus total protein bands.
protease and phosphatase inhibitors (50 mM Tris-HCl pH 8.0,
NaCl 150 mM, ND-40 0.5%, EDTA 1 mM, PMSF 1 mM, NaF
10 mM, Na3VO4 1 mM, b-glycerophosphate 25 mM). Beads Statistical Analysis
were washed at least six times with ice-cold lyses buffer and the Unless stated otherwise, all normalized band intensity data were
bound proteins were detected by Western blotting. statistically analyzed by students t test assuming equal variance using
MicrosoftH Excel software. The variance patterns in each set of
For Co-IP experiments, 25 mg of antibody was added to 100
data were previously checked by ANOVA from Excel data
500 mg of cell lysate and incubated (4 hr at 4uC) and then 100 ml
analysis tool package.
of equilibrated 50% protein-G Sepharose beads (Amersham
Pharmacia BiotechH) were added to the protein-antibody immune
complexes and incubated (1 h at 4uC). Beads were washed (66) Supporting Information
with lyses buffer and after the final wash, beads were suspended in Coordinate S1 Apoptin structure coordinates.
SDS sample buffer and resolved on SDS-PAGE gel, and the Co- (TXT)
IPed protein was detected by Western blotting. Other Western
Coordinate S2 Apoptin structure coordinates.
blotting experiments were performed as previously reported
(TXT)
[72,73,74]. Briefly, about 30 mg protein lysates were resolved by
SDS PAGE, transferred to PVDF-membrane (Amersham Bios- Coordinate S3 Apoptin structure coordinates.
ciencesH), membrane blocked (5% non-fat dry milk powder/tris- (TXT)
buffered saline with 0.25% v/v Tween-20 5% bovine serum Coordinate S4 Coordinates of Apoptin docking to
albumin, BSA). Membranes were washed and incubated with an BcrAbl.
appropriate secondary antibody conjugated with horseradish (TXT)
peroxidase (HRP) for 45 min at room temp and detected by
using enhanced chemiluminescent (ECL) staining (Amersham Coordinate S5 Coordinates of Apoptin docking to
BiosciencesH). BcrAbl.
(TXT)
Immunocytochemistry and fluorescent imaging Coordinate S6 Coordinates of Apoptin docking to
Cells were allowed to attach overnight, transfected with BcrAbl.
appropriate plasmids and after 1618 h of incubation cells were (TXT)
collected, washed with PBS, and fixed in 4% w/v paraformalde-
hyde/PBS. Thin smear of cells was prepared on standard Acknowledgments
microscope glass slides and air-dried. Cells were permeabilized
SP gratefully acknowledges Dr. Janice G. Dodd, Professor and Department
(0.1% triton X-100/PBS), blocked (5% BSA/PBS, 1 h) and Head, Physiology, University of Manitoba, for her advice and mentorship
incubated overnight at 4uC with an appropriate primary antibody, support during his PhD studies.
followed by incubation with Cy3 or FITC conjugated secondary
antibody. Slides were mounted with VectashieldH containing Author Contributions
DAPI. Fluorescence signals were acquired using Zeiss fluorescent
Conceived and designed the experiments: SP JS SKM ML. Performed the
microscope and analyzed by Zeiss Axiovision (Version 3.1) experiments: SP JRJ SM SKM. Analyzed the data: SP JS SKM ML.
software. Contributed reagents/materials/analysis tools: ML SKM. Wrote the
paper: SP JS SKM ML.

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