Computer-aided Molecular Design - Yuan-Ping Pang

Zinc Protein Simulations Using the Cationic Dummy Atom (CaDA) Approach

Amber all-atom force field parameters for the CaDA approach using an explicit solvent model

Table 1. The Most Recent Amber Force Field Parameters for Tetrahedron-Shaped Zinc Divalent Cation Used by the Cationic Dummy Atom (CaDA) Approach to Zinc Protein Simulations (Feb 25, 2005)

Table 2. Amber Atom Types and Charges of Histidinate, Hydroxide and Tetrahedron-Shaped Zinc Divalent Cation Used by the CaDA Approach

Generating Topology and Coordinates Files Using xLeap (AmberTools V1.5)
Parameter and example files:

• leaprc.zinc
• znb.lib
• hin.lib
• hydroxide.lib
• frcmod.zinc
• original_zinc.pdb
• revised_zinc.pdb

Download and copy the parameter files to respective directories (see commands below).

cp leaprc.zinc $AMBERHOME/dat/leap/cmd
cp znb.lib hin.lib hydroxide.lib $AMBERHOME/dat/leap/lib
cp frcmod.zinc $AMBERHOME/dat/parm

Launch xLeap by typing the following command in c shell of X11:
$AMBERHOME/exe/xleap –s –f $AMBERHOME/dat/leap/cmd/leaprc.ff99SB

Type the following commands in xLeap:
source $AMBERHOME/dat/leap/cmd/leaprc.zinc
m = loadpdb revised_zinc.pdb
addions m counterion 0
solvatebox m TIP3PBOX 10.0
saveamberparm m prmtop prmcrd

Notes:
xLeap does not recognize $AMBERHOME and you need to replace it with an actual path to the amber directory (e.g., /Applications/amber11).

"revised_zinc.pdb" is a pdb file that has been revised as follows:

Change the residue and atom names of the zinc divalent cation to ZNB and Zn, respectively. ZNB is the residue name of the tetrahedron-shaped zinc divalent cation and has 4 peripheral dummy atoms attached to the central zinc atom. xLeap will automatically add these dummies if they are not defined in the pdb file.

Change the residue name of the zinc-coordinating water molecule to HO- (hydroxide).

Change HIS and CYS to HIN and CYM, respectively, if these residues coordinate the zinc ion. HIN and CYM are anionic forms of histidine and cysteine, respectively.

Change GLU and ASP to GLH and ASH, respectively, if these residues form a hydrogen bond with HIN, HO-, or CYM. GLH and ASH are the neutral forms of glutamic and aspartic acids, respectively. Swap the coordinates of OE1 and OE2 of GLH or ASH if OE1 is closer to the hydrogen bond acceptor of HIN, HO-, or CYM than OE2 (see below).

"counterion" is either Na+ or Cl-.

"prmtop" and "prmcrd" are topology and coordinate files, respectively, for sander and/or pmemd

Ignore the "4n unknown element" message, wherein n is the number of the zinc ion in the protein, after "m = loadpdb revised_zinc.pdb." This benign message is caused by the lack of dummy atoms defined in the pdb file.

The CaDA Approach References

• Pang, Y.-P., Novel zinc protein molecular dynamics simulations: Steps toward antiangiogenesis for cancer treatment. J. Mol. Model. 1999, 5, 196-202.
• Pang, Y.-P.; Xu, K.; El Yazal, J.; Prendergast, F. G., Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach. Protein Sci. 2000, 9, 1857-1865.
• Pang, Y.-P., Successful molecular dynamics simulation of two zinc complexes bridged by a hydroxide in phosphotriesterase using the cationic dummy atom method. Proteins. 2001, 45, 183-189.
• Oelschlaeger, P.; Schmid, R. D.; Pleiss, J., Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations. Protein Eng. 2003, 16, (5), 341-350.
• Oelschlaeger, P.; Schmid, R. D.; Pleiss, J., Modeling domino effects in enzymes: Molecular basis of the substrate specificity of the bacterial metallo-beta-lactamases IMP-1 and IMP-6. Biochemistry 2003, 42, (30), 8945-8956.
• Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A. JG Park, PC Sill, EF Makiyi, AT Garcia-Sosa, CB Millard, JJ Schmidt, YP Pang, Bioorg. Med. Chem., 2006, 14, 395-408.
• Computer-aided Lead Optimization: Improved Small-Molecule Inhibitor of the Zinc Endopeptidase of Botulinum Neurotoxin Serotype A. Jing Tang, Jewn Giew Park, Charles B. Millard, James J. Schmidt, and Yuan-Ping Pang, PLoS ONE, 2007, 2(8): e761.
• Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis-Based Computer-Aided Molecular Design. Yuan-Ping Pang, Anuradha Vummenthala, Rajesh K. Mishra, Jewn Giew Park, Shaohua Wang, Jon Davis, Charles B. Millard, and James J. Schmidt, PLoS ONE, 2009, 4(11): e7730.
• Small Molecules Showing Significant Protection of Mice against Botulinum Neurotoxin Serotype A. Yuan-Ping Pang, Jon Davis, Shaohua Wang, Jewn Giew Park, Madhusoodana P. Nambiar, J. Schmidt, and Charles B. Millard, PLoS ONE, 2010, 5(4): e10129.

Q & A

Q: Energy minimization failed independent of what zinc protein was used.

A: This is likely due to special characters in leaprc.zinc, hin.lib, znb.lib, hydroxide.lib, or frcmod.zinc. These characters are introduced after copying the files from one operating system to the other. If you initially download the files to a Windows system, you need to follow the procedure below to make these files work properly in a Unix or Linux system.

1. Download and save the .zip files to a windows system
2. Transfer the .zip files to a Unix or Linux system
3. Change the ".zip" extension to ".gz"
4. Unzip the .gz files with the "gunzip" command
5. Add ".lib" extension to the hin, znb, and hydroxide files
6. Add ".zinc" extension to the leaprc and frcmod files

Another possibility is that the residue or atom name is not labeled correctly (see PDF file).

Q: The four dummy atoms do not point towards their respective coordinates after energy minimization.

A: This happens when DIELC is not 1.0 as the CaCA parameters are developed for DIELC = 1.0.

Q: The hydroxide always fused with one of the dummy atoms leading to an infinite electrostatic energy, when minimizing a protein containing a hydroxide-bound, tetrahedron-shaped zinc divalent cation.

A: This happens when the atom type of the dummy atom is mistakenly defined as "H" as it should be "DZ."

Q: The xLeap procedure failed when using 2CAB.pdb (carbonic anhydrase form B). However, the same procedure worked when using 1G54.pdb (carbonic anhydrase II).

A: This problem is caused by the 4 lines after the zinc atom line in the 2CAB.pdb (see below). Simply delete these lines and the procedure should work with 2CAB.pdb as well.

Q: Can the CaDA parameters be used with the ff03 force field?

A: The CaDA parameters work with all AMBER all-atom force fields excluding implicit solvent models.