The document describes an investigation of ligand binding in breast cancer resistance protein (BCRP) to develop resistance using computational methods. The objectives were to develop a homology model of BCRP, compare it to experimental topology, understand protein dynamics through normal mode analysis, study the role of substrate specificity exhibiting amino acids in the BCRP channel, and analyze evolutionary relationships of ABCG2 protein members. Key findings included identifying nucleotide and ligand binding sites, modeling BCRP structure, and predicting transmembrane helices in the membrane binding domain.

1. INVESTIGATION OF LIGAND BINDING IN BREAST
CANCER RESISTANCE PROTEIN TOWARDS DEVELOPING
RESISTANCE USING COMPUTATIONAL METHODS
Suman A. Tripathi
Under the Guidance of
Dr. Ravi Gor Mr. S. Prasanth Kumar
St. Kabir Institute of Bioinformatics Laboratory,
Professional Studies (SKIPS) Applied Botany Centre (ABC),
Ahmedabad- 380 054. University School of Sciences
Gujarat University,
Ahmedabad- 380 009.

2. Objective of the Study
1) Develop a homology model of breast cancer resistance protein (BCRP)
1) Develop a homology model of breast cancer resistance protein (BCRP)
2) Comparison of structure model with experimentally known topology
2) Comparison of structure model with experimentally known topology
3) Understand the protein dynamics of BCRP through coarse-grained
3) Understand the protein dynamics of BCRP through coarse-grained
normal mode analysis
normal mode analysis
4) Study the role of substrate specificity exhibiting amino acid in the BCRP
4) Study the role of substrate specificity exhibiting amino acid in the BCRP
channel and its interaction with various synthetic and natural
channel and its interaction with various synthetic and natural
compounds
compounds
5) Evolutionary relationship of homologous group of ABCG2 protein
5) Evolutionary relationship of homologous group of ABCG2 protein
members
members

3. BCRP
Breast Cancer Resistance Protein(BCRP)
Breast Cancer Resistance Protein(BCRP)
••Amembrane-associated protein belongs to ATP-bonding cassette
A membrane-associated protein belongs to ATP-bonding cassette
sub-family G member 2 (ABCG2)
sub-family G member 2 (ABCG2)
••Encodedby ABCG2 gene in human
Encoded by ABCG2 gene in human
••A655 amino acid protein encoded by abcg2 gene located on chromosomes
A 655 amino acid protein encoded by abcg2 gene located on chromosomes
4q22.
4q22.
••Widelyspread in all organism from bacteria to mammalia
Widely spread in all organism from bacteria to mammalia
••Function
Function
••Transportof a wide variety of compounds through cell membranes
Transport of a wide variety of compounds through cell membranes
against concentration gradient with ATP hydrolysis as energy for the
against concentration gradient with ATP hydrolysis as energy for the
process of substrate translocation.
process of substrate translocation.
••Diversephysiological processes, including transporting drugs (xenobiotics)
Diverse physiological processes, including transporting drugs (xenobiotics)
or drug conjugates and excreting endogenous metabolites or physiological
or drug conjugates and excreting endogenous metabolites or physiological
substrates.
substrates.

4. BCRP
Expression
Expression
••Highlyexpressed in tissue barriers such as the colon, small intestine, blood-
Highly expressed in tissue barriers such as the colon, small intestine, blood-
brain barrier (BBB), placenta and liver canalicular membrane.
brain barrier (BBB), placenta and liver canalicular membrane.
Structure
Structure
••BCRPis a half ABC transporter with one NBD followed by one TMD
BCRP is a half ABC transporter with one NBD followed by one TMD
••TMDof BCRP consists of 6 TM α-helices
TMD of BCRP consists of 6 TM α-helices
••NBDconstitute ATP binding site and MBD possess substrate binding site
NBD constitute ATP binding site and MBD possess substrate binding site
NBD-Nucleotide binding domain MSD-membrane spanning domain
NBD-Nucleotide binding domain MSD-membrane spanning domain
ECL- extracellular loop
ECL- extracellular loop

5. Role of BCRP in Drug Resistance
•In normal tissues, BCRP functions as a defense mechanism against toxins and
xenobiotics, with expression in the gut, bile canaliculi, placenta, blood-testis and
blood-brain barriers facilitating excretion and limiting absorption of potentially
toxic substrate molecules
•Including many cancer chemotherapeutic drugs. BCRP also plays a key role in
heme and folate homeostasis, which may help normal cells survive under
conditions of hypoxia.
•To play important roles in diverse physiological processes, including
transporting drugs (xenobiotics) or drug conjugates and excreting endogenous
metabolites or physiological substrates.

6. Methodology at a Glance

7. Hot Spots of BCRP
Domain
Domain Sequence range
Sequence range Descriptions
Descriptions
••NBD
NBD 1-286
1-286 ABC transporter domain
ABC transporter domain
••MBD
MBD 389-655
389-655 ABC transporter type-2
ABC transporter type-2
••Linkerregion
Linker region 287-388
287-388 Loop connecting 2 domains
Loop connecting 2 domains
Binding sites
Binding sites Sequence range
Sequence range Descriptions
Descriptions
••Nucleotide
Nucleotide 80-87
80-87 ATP binding site
ATP binding site
binding site
binding site
••Substrates
Substrates 482
482 Proximity of 10 Å was chosen to
Proximity of 10 Å was chosen to
binding site
binding site construct this site
construct this site

8. Consensus on Secondary
Structure
ATP binding site
based on annotated
entry of UniProtKB
Boundary of
NBD & linker
Boundary of
Linker & MBD
Glycosylation
motif
Substrate
binding site

9. Glycosylation motifs
Cont.
Motif
Motif Seq. span
Seq. span
NSSF
NSSF 338-341 (Linker region)
338-341 (Linker region)
NDST
NDST 418-421 (MBD)
418-421 (MBD)
NLTT
NLTT 557-560 (MBD)
557-560 (MBD)
NATG
NATG 596-599 (MBD)
596-599 (MBD)

10. Nucleotide and ligand
binding sites
ATP binding site
ATP binding site
••ATPbinding site found at 80-87 position
ATP binding site found at 80-87 position
in NBD domain
in NBD domain
••Constituteloop as its element
Constitute loop as its element
Substrate binding site
Substrate binding site
••Nucleotidebinding site was constructed
Nucleotide binding site was constructed
based on the potential role of Arg482 in
based on the potential role of Arg482 in
substrate specificity
substrate specificity
••Aminoacids found within 10 Å: Val445,
Amino acids found within 10 Å: Val445,
Lys452, Asp476, Leu478, Leu479, Pro480,
Lys452, Asp476, Leu478, Leu479, Pro480,
Met481, Arg482, Met483, Leu484, Pro485,
Met481, Arg482, Met483, Leu484, Pro485,
Ser486, Ile487, Ala505, Val508, Met509
Ser486, Ile487, Ala505, Val508, Met509
and Thr512.
and Thr512.

11. Closer look at ligand binding
sites
Arg482
Interfacing amino acids are equally distributed by TMH2, TMH3 and TMH4
Interfacing amino acids are equally distributed by TMH2, TMH3 and TMH4

12. Structure modeling of BCRP
Protein
••N-terminal (1-34) not modeled
N-terminal (1-34) not modeled
due to high confidence of
due to high confidence of
disorderness secured more than
disorderness secured more than
8 as loop element
8 as loop element
••Model with lowest eevalue
Model with lowest value
ranged from 1e-60 to 9.7e-32
ranged from 1e-60 to 9.7e-32
with 100% precision
with 100% precision
••NBD:9 alpha helices and 9
NBD: 9 alpha helices and 9
beta sheet
beta sheet
••Linker:4 helices
Linker: 4 helices
••MBD:6 TM helices
MBD: 6 TM helices
••ATPbinding site found
ATP binding site found
between 3rd alpha helix and 1stst
between 3rd alpha helix and 1
beta sheet.
beta sheet.

13. Trasmembrane helices prediction
at MBD using MBD domain with linker sequence by TMHMM 2.0
•TMH was predicted
•TMH was predicted using MBD domain with linker sequence by TMHMM 2.0
••Predicted 6 helices with plausible posterior probability
Predicted 6 helices with plausible posterior probability
••Probabilitywere all near to 11with exception 1 helical element wityh 0.7 between
Probability were all near to with exception 1 helical element wityh 0.7 between
fifth and sixth TM helices
fifth and sixth TM helices
••Goodcorrespondence between model with experimentally known membrane
Good correspondence between model with experimentally known membrane
topology
topology

14. Normal mode analysis Of
BCRP
••TheRMSD of displaced motion captured in frames
The RMSD of displaced motion captured in frames
were found to be linear with respect to 120 frames
were found to be linear with respect to 120 frames
considered in this study.
considered in this study.
••Allthe residues across the frames were found with
All the residues across the frames were found with
RSMD between 25.89 and 55.73 indicating
RSMD between 25.89 and 55.73 indicating
••nolarge atomic fluctuations.
no large atomic fluctuations.
••TheC-terminal region of MBD domain constituted
The C-terminal region of MBD domain constituted
with low modes of displacement.
with low modes of displacement.

15. Structure Validation
Errat graph indicating residues
Errat graph indicating residues
in the span of 40-80 in NBD are
in the span of 40-80 in NBD are
Ramachandran Plot
Ramachandran Plot mistraced . .Model reliable with
mistraced Model reliable with
(98.5 % favored model)
(98.5 % favored model) 87.263% overall quality
87.263% overall quality

16. Analysis of channel in MBD
domain
••Crucialamino acid Arg482 was found in channel pore
Crucial amino acid Arg482 was found in channel pore
••Porestatistics reveled that pore diameter with lowest diameter 1 Ȧ &
Pore statistics reveled that pore diameter with lowest diameter 1 Ȧ &
highest 24.3 Ȧ. .Shape identified as SUSDS (top to bottom)
highest 24.3 Ȧ Shape identified as SUSDS (top to bottom)

17. MSA and Phylogenetic
Analysis
Multiple
Multiple
Sequence
Sequence
Alignment
Alignment
(MSA) of
(MSA) of
ABCG2
ABCG2
protein
protein

18. MSA and Phylogenetic
Analysis

19. ATP interaction at NBD
domain
••Theinteraction profile was
The interaction profile was
encompassed with an overall
encompassed with an overall
energy of -74.69 KJ/mol with
energy of -74.69 KJ/mol with
other components such as H-
other components such as H-
bonding term of -20.54
bonding term of -20.54
KJ/mol and van der Waal
KJ/mol and van der Waal
interaction of -53.49 KJ/mol.
interaction of -53.49 KJ/mol.
••Thehydrogen bonding was
The hydrogen bonding was
observed with two residues,
observed with two residues,
Gly80 and Ser87 whereas the
Gly80 and Ser87 whereas the
whole ligand adopts the
whole ligand adopts the
geometrical fitting along the
geometrical fitting along the
predicted loop cavity element
predicted loop cavity element

20. Energetic contributions of
ligand set
Sr. No Compound Total Energy VDW H-Bond Elec
(KJ/mol) (KJ/mol) (KJ/mol) (KJ/mol)
Nucleotide binding site
1 ATP -31.558 -14.5271 -15.6002 -1.43081
Substrate binding site
2 Reserpine -103.723 -88.6627 -15.0604 0
3 VX 710 -98.8934 -75.8184 -23.0749 0
4 Nelfinavir -97.7238 -85.0661 -12.6577 0
5 Imatinib -97.6735 -88.824 -8.84946 0
6 CI1033 -95.0885 -88.7413 -6.34716 0
7 GF120918 -93.6321 -83.7334 -9.89869 0
8 Gefintinib -88.0638 -77.854 -10.2098 0
9 Tryprostatin A -83.1677 -78.1464 -5.02122 0
10 Ginsenoside 0
Rh 1 -83.1271 -72.6271 -10.5

21. Energetic contributions of
ligand set
11 Tamoxifen -82.4713 -82.4713 0 0
12 PPT -80.9421 -64.264 -16.6781 0
13 Ginsenoside Rg3 -80.1199 -67.1498 -12.9701 0
14 EKI 785 -78.7257 -68.284 -10.4417 0
15 Ginsenoside Rg1 -78.7094 -70.3318 -8.37763 0
16 Ginsenoside Rh 2 -77.6784 -77.6784 0 0
17 PPD -77.05 -63.1563 -13.8937 0
18 Omeprazole -76.556 -69.8996 -6.65648 0
19 Ko132 -75.2409 -65.4023 -9.8386 0
20 Ko143 -74.6726 -64.1726 -10.5 0
21 Ko134 -73.8305 -63.3305 -10.5 0
22 Saqinavir -72.0913 -65.0913 -7 0
23 Genistein -71.9116 -57.9335 -13.9781 0
24 Diethyl Stilbestrol -70.9086 -67.0059 -3.90275 0
25 17 Beta Estradiol -69.8645 -62.8645 -7 0
26 Fumitremorgin C -67.5392 -57.1165 -10.4227 0
27 Quercetin -66.1375 -43.4745 -22.6631 0
28 Estrone -63.5083 -54.4897 -9.01859 0
29 Ritonavir -50.4417 -45.6078 -4.83387 0

22. Small molecular docking at
MBD
Reserpine
Reserpine VX710
VX710 Nelfinavir
Nelfinavir
-103.723 KJ/mol
-103.723 KJ/mol -98.8934 KJ/mol
-98.8934 KJ/mol -97.7238 KJ/mol
-97.7238 KJ/mol
Imatinib
Imatinib Ginsenoside Rh1
Ginsenoside Rh1
-97.6735 KJ/mol
-97.6735 KJ/mol -83.1271 KJ/mol
-83.1271 KJ/mol

23. Heat map of Interaction
profiles

24. Major Outcomes
••Homology model with enhanced structural predictions with
Homology model with enhanced structural predictions with
comparison to previous reports.
comparison to previous reports.
••The normal mode analysis revealed that the NBD and MBD
The normal mode analysis revealed that the NBD and MBD
regions were fitted to small magnitude of atomic displacements
regions were fitted to small magnitude of atomic displacements
with exception of C-terminal region of MBD. Trajectory and
with exception of C-terminal region of MBD. Trajectory and
topology analysis yielded an averaged structure and indicated
topology analysis yielded an averaged structure and indicated
reliability
reliability
••The crucial role of Arg482 in substrate specificity was studied
The crucial role of Arg482 in substrate specificity was studied
using pore analysis and docking simulations indicating the
using pore analysis and docking simulations indicating the
potential role of aromatic linkers rather glycoside scaffold.
potential role of aromatic linkers rather glycoside scaffold.

25. Major Outcomes
••Binding modes of ATP in NBD binding site and phytochemical
Binding modes of ATP in NBD binding site and phytochemical
and synthetic compounds in MBD binding site.
and synthetic compounds in MBD binding site.
••Phylogenetic analysis confirmed the conservativeness among
Phylogenetic analysis confirmed the conservativeness among
different organisms and significant conservativeness at the
different organisms and significant conservativeness at the
binding sites.
binding sites.
••The glycosylation motifs were visually examined and correlated
The glycosylation motifs were visually examined and correlated
to the experimental topology.
to the experimental topology.
••We anticipate that this work will contribute a significant level
We anticipate that this work will contribute a significant level
towards the development of BCRP inhibitors and understand the
towards the development of BCRP inhibitors and understand the
mechanism of drug efflux.
mechanism of drug efflux.

26. Major Citations
Kimura Y, Morita S, Matsuo M, Ueda K, 2007. Mechanism of multidrug
recognition by MDR1/ABCB1. Cancer Sci., 9:1303-1310.
Borst P, Elferink RO, 2002. Mammalian ABC transporters in health and
disease. Annu. Rev. Biochem., 71: 537-592.
Xu J, Liu Y, Yang Y, Bates S, Zhang JT, 2004. Characterization of
oligomeric human half ABC transporter ABCG2/BCRP/MXR/ABCP in
plasma membranes. J.Biol. Chem. 279: 19781-19789.
Wang H, Lee EW, Zhou L, Mao Q, 2008. Membrane topology of the human
breast cancer resistance protein (BCRP/ABCG2) determined by epitope
insertion and Immunofluorescence. Biochem., 47: 13778–13787.
McDevitt CA, Collins RF, Conway M, Modok S, Storm J, Kerr ID, Ford
RC, Nuffield CR, 2006. Purification and 3D structural analysis of oligomeric
human multidrug transporter ABCG2. Structure., 14(11):1623-1632.

27. Thank you !!!