Characterizing binding site water molecules using
MD and FMO QM methods for rational drug design
Osamu Ichihara, Michelle Southey, Alexander Heifetz and Richard Law
Evotec (UK) Ltd, 114 Milton Park, Abingdon, OX14 4SA
Abstract
FMO Crystal Water Scoring: Thrombin Examples
The enthalpic stability of the crystallographically resolved water molecules in binding sites, as well as
solvating waters placed at the positions of high occupancy identified by molecular dynamics simulations,1)
were evaluated by the Fragment Molecular Orbital (FMO) method 2) using a neon atom as an explicit water
probe. The neon atom performed well as a spherical polarizable water probe providing the interaction
energy with the receptor. Solvating water molecules were classified into three classes of high, medium,
and low interaction energies revealing useful information on the hydration state of the binding site. The
classification criteria were validated using 285 thrombin and 165 Hsp90 ligand complexes published in the
PDB. Energetic profiles of binding site solvating water molecules obtained by FMO method will provide
highly valuable guidance for rational structure-based drug design.
 Preliminary binding site water classification criteria (red, amber, green traffic light system) were defined
by overlay analysis of the scored water molecules and 15 thrombin/ligand complex structures published
in PDB
 The results agree well with medicinal chemists perception of the thrombin catalytic site
Low int energy;
Medium int energy;
High int energy;
Readily replaced by hydrophobic group (int energy > -4.3kcal/mole)
Usually require a H-bonding motif to replace (int energy -4.3 to -8kcal/mole)
Hard to replace or can be replaced only with a very good H-bond motif (int energy < -8kcal/mole)
S4
Oxyanion hole
Use of FMO Method for Binding Site Water Characterization
 Interaction energies between crystallographically resolved binding site water molecules and
receptor/surrounding waters can be evaluated in terms of the sum of pair interaction energies (PIE) by
FMO
Key non-classical Hbond with Asp189 and
Gly219
 Hydrogen atoms need to be added; Only one of the H-bond network patterns among countless other
‘Unhappy’ water
possibilities can be evaluated by a single calculation
S1
with excess entropic
penalty in S1 pocket
Neon Atom as FMO QM Water Probe
 At around optimal H-bond distance water/methanol complex shows strong exchange repulsion
accompanied by equally strong electrostatic attraction (permanent and induced dipole) resulting in net
attraction
Figure 5: Thrombin ligand complex (PDB: 2ZNK)
overlaid with crystallographic water molecules
Figure 6: Thrombin ligand complex (PDB: 3EQ0)
overlaid with crystallographic water molecules
 Neon/methanol complex does not show such a strong electrostatic energy response (lack of permanent
Locating Ordered Solvating Waters by MD Simulation: Hsp90 Examples
dipole)
 2ns MD simulation using an apo Hsp90 structure generated from a Hsp90/ligand complex structure
water/methanol
(PDB: 2XJJ)
neon/methanol
 Nosé-Poincaré-Andersen (NPA) equations of motion, NVT ensemble, all the heavy atoms of protein
constrained, OPLS-AA/TIP3P forcefield used
 Cluster analysis of water oxygen positions from 2000 snapshots and positions of high water occupancy
were scored by FMO calculations
d
 Key conserved waters correctly positioned/scored
E (kcal)
E (kcal)
(HF + dispersion)
(electrostatic)
(exchange repulsion)
(charge transfer & mixed term)
Low int energy
Medium int energy
High int energy
Replacement of low-scoring water
with hydrophobic motif
(dispersion)
Replacement of medium
water with H-bond motif
d
Water replaced
by neon
Ordered water
d (Å)
d (Å)
Figure 1: EDA analysis of H-bond pairs (water/methanol and neon/methanol)
 Interaction energy profile of H-bond pairs involving water molecules can be mimicked using neon as a
spherical polarizable water probe and employing an interaction energy term (EHF+ Edisp – Eex)
Conserved waters
(orange: crystal water)
Figure 7: FMO scoring of MD-generated solvating water
molecules in Hsp90 binding site
 No need for calculating multiple water positions
Figure 8: Hsp90 ligand complex (PDB: 2XAB) overlaid with
MD-generated solvating water molecules
FMO Solvating Water Scoring: Validation in Thrombin and Hsp90
 285 Thrombin and 165 Hsp90 ligands whose complex structures are published in PDB were overlaid
with MD-generated solvating water molecules in the binding sites
 Atom types of the heavy atoms of ligand molecules within 1 Å distance from solvating water molecules
were examined
E (kcal)
d
E (kcal)
d
 Solvating water molecules are clearly separated into two groups, group A – waters predominantly
replaced by polar atoms (N, O), group B – waters predominantly replaced by hydrophobic atoms (FMO
interaction energy threshold of ca. -4.3kcal/mole as set in the previous section)
d
d
Water replaced
by neon
Water replaced
by neon
d (Å)
d (Å)
Figure 2: H-bond pair interaction energy: Water/Neon as an
acceptor
Figure 3: H-bond pair interaction energy: Water/Neon as a
donor
FMO for Binding Site Water Scoring: Procedure
2. Cut out the
area of interest
1. High resolution structure of
target protein with crystal waters
PDB: 3R3G (thrombin)
neon
(fragment)
Figure 9: FMO scoring of MD-generated solvating water molecules and atom type statistics of the ligands which replace them
(solvation water molecules are shown as red spheres)
References and Notes
3. Water replaced with neon and
interaction energy with protein
calculated (sum of all PIE)
1. For an example of locating sovating waters by MD, see; T. Young, R. Abel, B. Kim, B. J. Berne, and R. A. Friesner, PNAS, 104, 808 (2007)
2. K. Kitaura, E. Ikeo, T. Asada, T. Nakano, M. Uebayasi, Chem. Phys. Lett. 313, 701 (1999), D. G. Fedorov, R. M. Olson, K. Kitaura, M. S. Gordon, S. Koseki,
J. Comput. Chem., 25, 872 (2004) 872.
3. All FMO calculations were performed at MP2/6-31G* theory level using GAMESS QM suite; M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S.
Gordon, J. H. Jensen, S. Koseki, N. Matsunaga, K. A. Nguyen, S. Su, T. L. Windus, M. Dupuis, J. A. Montgomery, J. Comput. Chem., 14, 1347 (1993)
Figure 4: FMO binding site water scoring : Procedures
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