See also off_frcmod.zip for non standard parameters for Magnesium, ATP and ligand.
=== Setup description ===
The crystallographic structure of human 3-phosphoinositide-dependent protein kinase 1 (PDK1) with allosteric ligand 1F8 was obtained from the PDB databank(REF1) (PDB ID 3ORX). Residues 75 to 359 were included, however residue 237 was not resolved in the crystal structure. All crystallographic waters and ions were removed, and the protein coordinates were used as an input for homology modelling with the software MODELLER(REF2) along with the amino acid sequence for residues 75 – 359. The resulting model protein was capped with both N-methyl amide and acetyl groups at the C and N termini respectively. Histidine residues were modelled as the neutral ε-protonated tautomer and phosphoserine241 was modelled with a single protonated phosphate group. Parameters for phosphoserine were provided by Sticht et al.(REF3)
The substrate of PDK1 is a 16-mer peptide, which was truncated and modelled as sequence KTFAGT, and capped with an N-methyl amide group at the C terminus. Coordinates for this peptide or other substrates of PDK1 were not available, and so predicted poses were obtained using the software Pepsite2.(REF4) The ligand 1F8 is covalently attached to the allosteric pocket at Cys148. Parameters were adapted for this bond by extracting coordinates from the crystal structure for the allosteric ligand, along with the Sγ and Cδ atoms of Cys148, and Cδ was replaced with a hydrogen atom. For the resulting thio-ether, partial charges could then be derived using the AM1-BCC methodology. The partial charges initially assigned were modified to replicate the bond of a bound cysteine residue as defined in the Amber force field. The charge on the ligand could then be balanced to give an overall neutral charge. Atoms belonging to the protein were then removed, and the coordinates of the ligand were aligned to the X-ray crystal structure of the protein ligand complex.
Coordinates for ATP were obtained from PDB ID 4AW0, as ATP was not present in 3ORX. ATP parameters from Carslon et al.(REF5) were used. Two magesium ions are present in the ATP binding site, for which an octahedral dummy model was used, as developed by Kamerlin et al.(REF6) Water molecules were added manually in order for octahedral coordination to be obtained at each Mg2+ centre. Counter-ions were added using xleap from the Amber14 software package, in order to neutralise the system. General Amber Force Field parameters(REF7) were assigned to ligand atoms, while the FF14SB-ILDN force field(REF8) was used to describe the protein. Counter-ions were described using parameters developed by Joung & Cheatham.(REF9)
1 Sadowsky, J. D. et al. Proc. Natl. Acad. Sci. 2011, 108 (15), 6056–6061.
2 Sali, A. Mol. Med. Today, 1995, 1 (6), 270–277.
3 Homeyer, N. et al., J. Mol. Model. 2006, 12, 281-289.
4 Trabuco L. G., et al., Nucleic Acids Res. 2012, 40(Web Server issue):W423-426.
5 Meagher, K. L. et al., Journal of Computational Chemistry 2003, 24, 1016
6 Kamerlin, S. C. L. et al., J. Phys. Chem. B., 2014, 118 (16), 4351–4362.
7 Case, D.A. et al., The Amber14 Software Package. University of California, San Francisco. 2015.
8 Maier, J. A. et al., J. Chem. Theory Comput. 2015, 11, 3696–3713.
9 Joung, I. S. & Cheatham, T. E., J. Phys. Chem. B, 2008, 112, 9020–90
=== Attachment === Input: protein_raw_3orx.pdb
Output: Protein.pdb Peptide.pdb 1F8.pdb ATP.pdb
See also off_frcmod.zip for non standard parameters for Magnesium, ATP and ligand.
=== Setup description === The crystallographic structure of human 3-phosphoinositide-dependent protein kinase 1 (PDK1) with allosteric ligand 1F8 was obtained from the PDB databank(REF1) (PDB ID 3ORX). Residues 75 to 359 were included, however residue 237 was not resolved in the crystal structure. All crystallographic waters and ions were removed, and the protein coordinates were used as an input for homology modelling with the software MODELLER(REF2) along with the amino acid sequence for residues 75 – 359. The resulting model protein was capped with both N-methyl amide and acetyl groups at the C and N termini respectively. Histidine residues were modelled as the neutral ε-protonated tautomer and phosphoserine241 was modelled with a single protonated phosphate group. Parameters for phosphoserine were provided by Sticht et al.(REF3)
The substrate of PDK1 is a 16-mer peptide, which was truncated and modelled as sequence KTFAGT, and capped with an N-methyl amide group at the C terminus. Coordinates for this peptide or other substrates of PDK1 were not available, and so predicted poses were obtained using the software Pepsite2.(REF4) The ligand 1F8 is covalently attached to the allosteric pocket at Cys148. Parameters were adapted for this bond by extracting coordinates from the crystal structure for the allosteric ligand, along with the Sγ and Cδ atoms of Cys148, and Cδ was replaced with a hydrogen atom. For the resulting thio-ether, partial charges could then be derived using the AM1-BCC methodology. The partial charges initially assigned were modified to replicate the bond of a bound cysteine residue as defined in the Amber force field. The charge on the ligand could then be balanced to give an overall neutral charge. Atoms belonging to the protein were then removed, and the coordinates of the ligand were aligned to the X-ray crystal structure of the protein ligand complex.
Coordinates for ATP were obtained from PDB ID 4AW0, as ATP was not present in 3ORX. ATP parameters from Carslon et al.(REF5) were used. Two magesium ions are present in the ATP binding site, for which an octahedral dummy model was used, as developed by Kamerlin et al.(REF6) Water molecules were added manually in order for octahedral coordination to be obtained at each Mg2+ centre. Counter-ions were added using xleap from the Amber14 software package, in order to neutralise the system. General Amber Force Field parameters(REF7) were assigned to ligand atoms, while the FF14SB-ILDN force field(REF8) was used to describe the protein. Counter-ions were described using parameters developed by Joung & Cheatham.(REF9)
1 Sadowsky, J. D. et al. Proc. Natl. Acad. Sci. 2011, 108 (15), 6056–6061. 2 Sali, A. Mol. Med. Today, 1995, 1 (6), 270–277. 3 Homeyer, N. et al., J. Mol. Model. 2006, 12, 281-289. 4 Trabuco L. G., et al., Nucleic Acids Res. 2012, 40(Web Server issue):W423-426. 5 Meagher, K. L. et al., Journal of Computational Chemistry 2003, 24, 1016 6 Kamerlin, S. C. L. et al., J. Phys. Chem. B., 2014, 118 (16), 4351–4362. 7 Case, D.A. et al., The Amber14 Software Package. University of California, San Francisco. 2015. 8 Maier, J. A. et al., J. Chem. Theory Comput. 2015, 11, 3696–3713. 9 Joung, I. S. & Cheatham, T. E., J. Phys. Chem. B, 2008, 112, 9020–90
PDK1.zip
off_frcmod.zip