Zinc Protein Simulations

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

Procedure

1. Replace the zinc divalent cation (one-atom representation) with the tetrahedron-shaped zinc divalent cation (five-atom representation)
2. Deprotonate the zinc ligands (use histidinate for histidine, hydroxide for water, CYM for cysteine)
3. Minimize the tetrahedron-shaped zinc divalent cation with a positional constraint applied to the protein.
4. Minimize the tetrahedron-shaped zinc divalent cation and the zinc ligands with a positional constraint applied to the rest of the protein.
5. Minimize the entire complex and start MD simulations without any restraint or constraint.

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.

Frequently Asked Questions

Question 1:

Following the generation of input files to Sander using the xLeap procedure described at the zinc protein simulation website, an energy minimization failed independent of what zinc protein was used. What went wrong?

Answer 1:

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

It is possible that the cysteine coordinates are mistakenly assigned as “CYX;” they should be assigned as “CYM.”

Another possibility is that the zinc cation is labeled incorrectly in the pdb file of the zinc protein (see below).

Question 2:

The four dummy atoms do not point towards their respective coordinates after an energy minimization. What went wrong?

Answer 2:

The CaCA parameters are developed for DIELC = 1.0 and work improperly when DIELC is not 1.0.

Question 3:

In an energy minimization of a protein with a hydroxide-bound, tetrahedron-shaped zinc divalent cation, the hydroxide always fused with one of the dummy atoms leading to an infinite electrostatic energy. What went wrong?

Answer 3:

This happens when the atom type of the dummy atom is mistakenly defined as “H;” it should be defined as “DZ.”

Question 4:

The xLeap procedure to generate input files to Sander failed when using 2CAB.pdb (carbonic anhydrase form B). However, the same procedure worked when using 1G54.pdb (carbonic anhydrase II). Why?

Answer 4:

The zinc atom is bonded to three histidines in 2CAB.pdb. Lines 2172–2175 in 2CAB.pdb should to be deleted to avoid adding bonds between zinc and its coordinates. In addition, lines 717 and 2170 in 2CAB.pdb should be deleted to avoid incompatibility issues with Gln74 and Phe260.

Question 5:

Does it matter if the ff99 or ff03 force field is used in xLeap with the .lib and frcmod files?

Answer 5:

The result should be the same whether you use the ff99 or ff03 force field.

Simulating Zinc Proteins Using the CaDA Approach: Generating Topology and Coordinates Files as Input into Sander Using xLeap

1. Change the residue name of the histidine that coordinates the zinc ion to "HIN" (histidinate, the anionic form of histidine), see zinc.pdb below
2. Change the residue name of the glutamic or aspartic acid that has a hydrogen bond to the zinc-bound histidine to "GLH" or "ASH" (the neutral form of glutamic or aspartic acid)
3. Change the residue name of the cysteine that coordinates the zinc ion to "CYM" (the anionic form of cysteine)
4. Change the residue name of the water molecule that coordinates the zinc ion to "HO-" (hydroxide), see zinc.pdb below
5. Change the residue and atom names of the zinc ion to "ZNB" (the tetrahedron-shaped zinc divalent cation) and "Zn", respectively; the coordinates of the four dummy atoms attached to the central zinc ion are not available from the crystal structure, xLeap will automatically add four dummy atoms to the central zinc ion once xLeap is loaded with znb.lib, see zinc.pdb below
6. Copy "znb.lib," "hin.lib," "hydroxide.lib," and "frcmod.zinc" (see below) to the directory where you want to launch xLeap. The formatting of the force-field or library file should be preserved as is or xLeap will not recognize it as library or force- field file.
7. Type $AMBERHOME/exe/xleap and the following commands (except comments in parentheses) within xLeap
   • source leaprc.ff99 (load the amber force field)
   • loadoff znb.lib (load the tetrahedron-shaped zinc divalent cation)
   • loadoff hin.lib (load the histidinate as a zinc coordinate)
   • loadoff hydroxide.lib (load the hydroxide as a zinc coordinate)
   • loadamberparams frcmod.zinc (load the force field parameters for the tetrahedron-shaped zinc divalent cation)
   • zinc = loadpdb zinc.pdb (load the zinc protein coordinates)
   • saveamberparm zinc prmtop prmcrd (prmtop and prmcrd are topology and coordinate files, respectively)
8. 
   

   Related Files

   • znb.lib
   • hin.lib
   • hydroxide.lib
   • frcmod.zinc
   • zinc.pdb