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

Assisted Model Building with Energy Refinement (AMBER) all-atom force field parameters for the CaDA method using an explicit solvent model

Bond K [kcal/(mol Å2)] Req (Å)
DZ-ZN 640 0.90
DZ-DZ 640 1.470
Angle K [kcal/(mol radian2)] Teq (deg.)
DZ-ZN-DZ 55 109.50
DZ-DZ-DZ 55 60.0
DZ-DZ-ZN 55 35.25
Dihedral IDIVF Vn/2 (kcal/mol) γ(deg.) N
ZN-DZ-DZ-DZ 1 0 35.3 2
DZ-ZN-DZ-DZ 1 0 120.0 2
DZ-DZ-DZ-DZ 1 0 70.5 2
VDW mass r*(Å) Eps (kcal/mol) VDW mass r*(Å) Eps (kcal/mol)
ZN 53.38 3.1 1E-6 DZ 3.0 0 0

AMBER atom types and charges of the histidinate, hydroxide and tetrahedron-shaped zinc divalent cation used by the CaDA method

Atom name Atom type Charge
N N -0.5641
H H 0.2469
CA CT 0.3171
HA H1 0.0096
CB CT -0.1347
HB2 HC 0.0083
HB3 HC 0.0381
CG CC 0.1504
ND1 NA -0.7626
CE1 CR 0.4994
HE1 H5 -0.0295
NE2 NB -0.7656
CD2 CV 0.0405
HD2 H4 0.0525
C C 0.4588
O O -0.5653
H1 HO 0.2049
O OH -1.2049
Tetrahedron-shaped zinc divalent cation
DZ DZ 0.5

Generating topology and coordinates files using xLeap (AmberTools V1.5)

Parameter and example files:

Note: If downloading files using Safari, the filename extensions may need to be manually changed from ".dms" to ".txt" before the files can be opened.

Download and copy the parameter files to the 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/leap/parm

Launch xLeap by typing the following command in the 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

xLeap does not recognize $AMBERHOME and you need to replace it with an actual path to the amber directory (for example, /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 which has four 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 acid and aspartic acid, 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).

ATOM 1622 OE1 GLU C 94 30.263 40.009 33.982 1.00 31.93 O
ATOM 1623 OE2 GLU C 94 30.708 38.077 34.933 1.00 33.98 O1-
ATOM 1622 OE1 GLH C 94 30.708 38.077 34.933 1.00 33.98 O
ATOM 1623 OE2 GLH C 94 30.263 40.009 33.982 1.00 31.93 O1-

"counterion" is either Na+ or Cl-.

"prmtop" and "prmcrd" are topology and coordinate files, respectively, for sander and 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 method references

  • Pang YP, Davis J, Wang S, Park JG, Nambiar MP, Schmidt JJ, Millard CB. Small molecules showing significant protection of mice against botulinum neurotoxin serotype A. PLOS One. 2010;5:e10129.
  • Pang YP, Vummenthala A, Mishra RK, Park JG, Wang S, Davis J, Millard CB, Schmidt JJ. Potent new small-molecule inhibitor of botulinum neurotoxin serotype A endopeptidase developed by synthesis-based computer-aided molecular design. PLOS One. 2009;4:e7730.
  • Tang J, Park JG, Millard CB, Schmidt JJ, Pang YP. Computer-aided lead optimization: Improved small-molecule inhibitor of the zinc endopeptidase of botulinum neurotoxin serotype A. PLOS One. 2007;2: e761.
  • Park JG, Sill PC, Makiyi EF, Garcia-Sosa AT, Millard CB, Schmidt JJ, Pang YP. Serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype A. Bioorganic Medicinal Chemistry. 2006;14:395.
  • Oelschlaeger P, Schmid RD, 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: 8945.
  • Oelschlaeger P, Schmid RD, Pleiss J. Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations. Protein Engineering, Design and Selection. 2003;16:341.
  • Pang YP. Successful molecular dynamics simulation of two zinc complexes bridged by a hydroxide in phosphotriesterase using the cationic dummy atom method. Proteins. 2001;45:183.
  • Pang YP, Xu K, El Yazal J, Prendergast FG. Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach. Protein Science. 2000;9:1857.
  • Pang YP. Novel zinc protein molecular dynamics simulations: Steps toward antiangiogenesis for cancer treatment. Journal of Molecular Modeling. 1999; 5:196.

Questions and answers

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 another. 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 the ".lib" extension to the hin, znb, and hydroxide files.
  6. Add the ".zinc" extension to the leaprc and frcmod files.

Another possibility is that the residue or atom name is not labeled correctly.

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

A: This happens when DIELC is not 1.0 because the CaDA 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" when 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 four lines after the zinc atom line in the 2CAB.pdb (see Figure 1). 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.

Q: Is the CaDA method applicable to simulation of a protein with a five-ligand zinc divalent ion?

A: Yes. Typically, a five-ligand zinc divalent ion stems from zinc coordination with four ligands, one of which is an ambidentate ligand.