Simulating c-Src

Srija Makkapati
c-Src and Imatinib
- c-Src is a tyrosine kinase protein that can
cause uncontrolled cell growth.
- Thus, c-Src is the cause of many types of
cancer, including colon, liver, and lung
cancer.
- c-Src is an enzyme which phosphorylates
other proteins, destabilizing the protein and
preventing it from causing rapid cell growth.
- The drug Imatinib is very effective on other
tyrosine kinases such as c-Abl and c-kit, in
contrast to c-Src.
- This is surprising because c-Src is a close
relative of c-Abl and c-kit.
Simulation Overview
- Obtained PDB file of c-Src complexed with Imatinib
with PDB ID 2OIQ
- Removed drug from protein-drug system and
added enough salt to create a 0.15 M salt solution
using xLEaP
- Placed protein (containing 80351 atoms) in a
truncated octahedron shaped box with 23,945
explicit water molecules with AMBER 14
- Minimized and heated system to temperature of
300 K (heating process lasted approximately 50
ps)
- Ran 1 ns MD simulation on the heated system at a
speed of 0.11 ns/day, took a total of 9 days to
complete
- Analyzed results using CPPTRAJ and created
temperature, potential energy, RMSD, and RMSF
plots for the simulation
- Temperature increased
approximately linearly from 0 to 50
ps, and stabilized at 300 K.

- Temperature fluctuates around 5 K.

- Potential energy increased
approximately linearly from 0
to 50 ps, peaked at 50 ps,
and stabilized at around
-2.441 ⋅105 kcal/mol by 200
ps.

- Potential energy fluctuates

about 1000 kcal/mol.
- The RMSD plot for 0 to 600 ps
(not shown) displayed large
fluctuations up to 1.5
angstroms.

- RMSD begins to stabilize

around 900 ps, fluctuating
about 0.25 angstroms.

- However, the RMSD

fluctuations increase at 1000
ps, indicating that the protein is
unfolding again.

- Running a longer simulation

would result in a more stable
RMSD plot and a more stable
protein structure.
- The majority of the RMSF plot
is relatively stable, fluctuating
about 1 angstrom.

- The RMSF plot has two main

points of interest.

- At around residue 270, the

RMSF spikes, most likely due
to the presence of the P-loop.

- At around residues 410-425,

the RMSF spikes notably
because the activation loop is
located at those residues.
- The average distance is
approximately 7
Angstroms.

- The distance has not

stabilized fully, indicating
again that the protein has
not found a stable
structure.

- Distance fluctuates around

1 Angstrom.
c-Src Structure

DFG motif
Imatinib

Activation loop
P loop
Drug Binding Site

Met 341
Imatinib

Tyr 340

Thr 338
Met 314
ATP Binding Site

Binding site
Skills Acquired
- Gained proficiency in use of the command line
- Learned the basic structure of a protein
- Gained experience with visualizing proteins and displaying RMSD plots in
VMD
- Learned to use xLEaP to prepare proteins for simulations (i.e. add salt, add
water box)
- Gained experience with using AMBER to run simulations and CPPTRAJ to
analyze results
- Learned to create plots with xmgrace
Conclusions
- c-Src is an enzyme which can cause cancer if a gene is mutated or is not
controlled by phosphorylating proteins
- Simulations of c-Src were run under relatively stable conditions (i.e.
temperature stabilized at 300 K, pressure stabilized at 1 atm)
- Simulations showed a high RMSF at the P loop and activation loop
- Further investigation may involve running simulations longer to allow RMSD
and distance between activation loop and ATP binding site to settle, and allow
the protein to find a more stable structure
References
D.A. Case, T.A. Darden, T.E. Cheatham, III, C.L. Simmerling, J. Wang, R.E. Duke, R. Luo, R.C. Walker, W. Zhang, K.M. Merz, B.
Roberts, B. Wang, S. Hayik, A. Roitberg, G. Seabra, I. Kolossváry, K.F. Wong, F. Paesani, J. Vanicek, J. Liu, X. Wu, S.R.
Brozell, T. Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang, M.-J. Hsieh, G. Cui, D.R. Roe, D.H. Mathews, M.G. Seetin, C.
Sagui, V. Babin, T. Luchko, S. Gusarov, A. Kovalenko, and P.A. Kollman (2010), AMBER 14, University of California, San
Francisco.

PDB ID: 2OIQ

Seeliger, M.A., Nagar, B., Frank, F., Cao, X., Henderson, M.N., Kuriyan, J. (2007) c-Src Binds to the Cancer Drug Imatinib with
an Inactive Abl/c-Kit Conformation and a Distributed Thermodynamic Penalty. doi:10.2210/pdb2oiq/pdb

Nagar, B., Hantschel, O., Young, M., Scheffzek, K., Veach, D., Bornmann, W., . . . Kuriyan, J. (2003). Structural basis for the
auto-inhibition of c-Abl tyrosine kinase. Cell, 112, 859-871. doi:10.2210/pdb1opl/pdb

Seeliger, M. A., Nagar, B., Frank, F., Cao, X., Henderson, M. N., & Kuriyan, J. (2007). C-Src Binds to the Cancer Drug Imatinib with an
Inactive Abl/c-Kit Conformation and a Distributed Thermodynamic Penalty. Cell, 15, 299-311. doi:10.1016/j.str.2007.01.015