The document describes the waterswap method developed by Christopher Woods for calculating absolute protein-ligand binding free energies using molecular dynamics simulations. Waterswap uses a thermodynamic integration approach to swap a ligand and water cluster between a protein box and a water box. This allows a direct calculation of the binding free energy. The method was tested on thrombin-ligand complexes and showed reasonable agreement with experimental binding affinities. Key advantages of waterswap include its ability to decompose binding free energies and provide insights into residue-level contributions and the effects of ligand hydrophobicity.

1. Using waterswap to predict and
understand binding affinities
Christopher Woods

2. Introduction
• Developer of software and algorithms to
predict protein-ligand binding free energies
• Binding free energy measures binding
affinity, can be directly related to Ki
• Developed “waterswap”. First-principles,
calculation of absolute binding free energies

3. Protein
Ligand
+

4. Protein
Ligand
Complex

5. Protein
Ligand
Complex
ΔGbind

6. Protein
Ligand
Complex
ΔGbind

7. Biochemistry occurs in the aqueous phase

8. Protein(aq)Ligand(aq)

9. Protein(aq)Ligand(aq)
WaterComplex(aq)

10. Protein(aq)Ligand(aq)
WaterComplex(aq)
ΔGbind

11. Protein(aq)Ligand(aq)
WaterComplex(aq)
ΔGbind

12. Protein(aq)Ligand(aq)
WaterComplex(aq)
ΔGbind

13. Protein(aq)Ligand(aq)
WaterComplex(aq)
ΔGbind

14. • Woods,&J&Chem&Phys,&Vol&134,&p054114,&2011&
• &h7p://dx.doi.org/10.1063/1.3519057&
Waterswap&Method&
&
Uses&the&fact&that&
proteinJligand&binding&is&
really&a&compeLLon&
between&the&ligand&and&
water&for&binding&to&the&
protein&

15. Protein:Water(aq)Ligand(aq)

16. Protein:Water(aq)Ligand(aq)

17. Protein:Water(aq)Ligand(aq)
Water(aq)Protein:Ligand(aq)

18. Protein:Water(aq)Ligand(aq)
Water(aq)Protein:Ligand(aq)
ΔGbind

19. Protein:Water(aq)Ligand(aq)
Water(aq)Protein:Ligand(aq)
ΔGbind
(*)

20. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box

21. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box

22. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]

23. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
λ=0.0

24. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
λ=0.0
100%
0%

25. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
λ=0.2
80%
20%

26. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
λ=0.5
50%
50%

27. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
λ=0.8
20%
80%

28. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
λ=1.0
0%
100%

29. Perform Thermodynamic Integration (TI) along the
Waterswap λ coordinate.This results, directly,
in the absolute binding free energy
!20$
!18$
!16$
!14$
!12$
!10$
!8$
!6$
!4$
!2$
0$
0.0$ 0.2$ 0.4$ 0.6$ 0.8$ 1.0$
Free$Energy$/$kcal$mol01$
λ$

30. Perform Thermodynamic Integration (TI) along the
Waterswap λ coordinate.This results, directly,
in the absolute binding free energy
!20$
!18$
!16$
!14$
!12$
!10$
!8$
!6$
!4$
!2$
0$
0.0$ 0.2$ 0.4$ 0.6$ 0.8$ 1.0$
Free$Energy$/$kcal$mol01$
λ$

31. Perform Thermodynamic Integration (TI) along the
Waterswap λ coordinate.This results, directly,
in the absolute binding free energy
!20$
!18$
!16$
!14$
!12$
!10$
!8$
!6$
!4$
!2$
0$
0.0$ 0.2$ 0.4$ 0.6$ 0.8$ 1.0$
Free$Energy$/$kcal$mol01$
λ$
ΔGbind

32. How to use Waterswap?

33. Waterswap
is built into
Sire, available
from
http://siremol.org

34. Sire can be
downloaded using
the links on
this site...

35. ...and there are
full instructions
on how to use
waterswap

52. …but,
• waterswap is easy to use…
• …but setting up a protein-ligand complex
for simulation requires expert knowledge
and is not trivial
• waterswap results depend on the quality of
the input model

53. Test Application to
Thrombin

54. Cl
NHO
N
R
O
CH3
H2
C
CH3
H2
C
C
H2
CH3
H2
C
H
C
CH3
CH3
H2
C
C
H2
CH
CH3
CH3
H2
C
H2
C
H2
C
C
H2
H2
C
C
H2
H2
C
C
H2
1
2
3
4
5
6
7
8
9
10
R

55. 20 30 40 50 60 70
Dynamics
Dynamics + Waterswap

56. 20 30 40 50 60 70
Dynamics
20.crd
20.top
Dynamics + Waterswap

57. 20 30 40 50 60 70
Dynamics
Waterswap
20.crd
20.top
Dynamics + Waterswap

58. 20 30 40 50 60 70
Dynamics
Waterswap
G20ns
20.crd
20.top
Dynamics + Waterswap

59. 20 30 40 50 60 70
Dynamics
Waterswap
G20ns
20.crd
20.top
30.crd
30.top
G30ns
Waterswap
Dynamics + Waterswap

60. 20 30 40 50 60 70
Dynamics
Waterswap
G20ns
20.crd
20.top
30.crd
30.top
G30ns
Waterswap
40.crd
40.top
50.crd
50.top
60.crd
60.top
70.crd
70.top
G40ns G50ns G60ns G70ns
Waterswap
Waterswap
Waterswap
Waterswap
Dynamics + Waterswap

61. 20 30 40 50 60 70
Dynamics
Waterswap
G20ns
20.crd
20.top
30.crd
30.top
G30ns
Waterswap
40.crd
40.top
50.crd
50.top
60.crd
60.top
70.crd
70.top
G40ns G50ns G60ns G70ns
Waterswap
Waterswap
Waterswap
Waterswap
Dynamics + Waterswap
< >

62. 20 30 40 50 60 70
Dynamics
Waterswap
G20ns
20.crd
20.top
30.crd
30.top
G30ns
Waterswap
40.crd
40.top
50.crd
50.top
60.crd
60.top
70.crd
70.top
G40ns G50ns G60ns G70ns
Waterswap
Waterswap
Waterswap
Waterswap
Dynamics + Waterswap
< >
Gbind

63. Cl
NHO
N
R
O
CH3
H2
C
CH3
H2
C
C
H2
CH3
H2
C
H
C
CH3
CH3
H2
C
C
H2
CH
CH3
CH3
H2
C
H2
C
H2
C
C
H2
H2
C
C
H2
H2
C
C
H2
1
2
3
4
5
6
7
8
9
10
R

64. -6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-3.5
-32 -30 -28 -26 -24 -22 -20
Experiment/kcalmol-1
Simulation / kcal mol-1
R2=0.82
1
23
4
108
5
6
79

65. !
Simulation should not try to
compete with experiment.
!
The job of simulation is to
provide inspiration and insight

66. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]

67. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]

68. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]

69. Free Energy Decomposition
• As we integrate the total waterswap
binding free energy...
• ...we also integrate free energy changes in
the “protein” box and the “water” box
• Result are free energies that tell you if a
ligand’s binding strength comes from a
natural affinity for the protein, or an
aversion to water

70. -6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-3.5
-10 -8 -6 -4 -2
Experiment/kcalmol-1
Simulation / kcal mol-1
R2=0.14
1
2
3
4
8 10
6
7
5
9
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-3.5
-26 -24 -22 -20 -18 -16 -14 -12
Experiment/kcalmol-1
Simulation / kcal mol-1
R2=0.84
1
23
4
5
6
108
79
Specificity driven by “water” box, i.e.
the more hydrophobic the ligand, the less it
wants to be in the water box, and the more it
wants to be in the protein box.
!
This shows that a “better” ligand is only better
because it is more hydrophobic
Protein Box Water Box

71. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]

72. Waterswap uses a λ-coordinate to swap a ligand and a
water cluster between a protein box and a water box
Protein Box Water Box
E = (1 )[Eprotein:cluster + Ewater:ligand]+
( )[Eprotein:ligand + Ewater:cluster]
Eresidue:cluster
Eresidue:ligand

73. Free Energy Decomposition
• As we integrate the total waterswap
binding free energy...
• ...we also integrate the individual
contributions from all of the binding site
residues
• Result is a “free energy” that indicates
whether the residue:ligand or
residue:water complex is more stable

74. Phe227Phe227

75. Asp189
Glu192
Glu192
p189

76. TotalElectrostatic
Phe227Phe227
Asp189
Glu192
Asp189
Glu192

77. ure S1. Experimentally measured binding affinities for the 10 ligands studied in thi
k (m-chlorobenzyl) and for the ten benzamidine analogs. Binding affinities are tak
m Muley et al., doi: 10.1021/jm9016416 (reference 32 in our paper).
8
10
-9
-8
-7
-6
-5
-4
-3
0 1 2 3 4 5 6 7 8 9 10
BindingAffinity/kcalmol-1
Ligand
m-chlorobenzyl
benzamidine
Replacing m-chlorobenzyl group with benzamidine
group systematically improves binding of the ligands

78. Conclusion
• Waterswap enables direct, first-principles
calculation of absolute binding free energies
• (but results depend on quality of model!)
• Free energies can be decomposed to per-
residue and per-water components
• Aim is to provide inspiration and insight

79. Appendix
• waterswap is just one of our tools…
• Also have ligandswap, which calculates
relative binding free energies by swapping
one ligand with another
• Also have waterview, that lets you quickly
visualise water dynamics in a binding site,
e.g.

82. Acknowledgements
• Organisers for inviting me and allowing me to talk
• You for your attention
• Dr. Maturos Malaisree (doing most of the work!)
• Dr. Julien Michel (discussions and providing thrombin test system)
• Prof.Adrian Mulholland, Simon McIntosh-Smith, Ben Long
• EPSRC and now BBSRC for funding
• eInfraStructureSouth for GPU compute
• ACRC (Bristol) for CPU compute
• Get the software at http://siremol.org
• Get in touch via Christopher.Woods@bristol.ac.uk

83. Identity Constraint
• How do we “identify” the cluster of water to be
swapped with the drug?
• We developed the identity constraint.This is a
new way of labelling water molecules in a
simulation that is based on where the molecule
is in space, rather than where it is located in the
input coordinate file.
• Allows definition of water clusters without
using restraints or external perturbations

92. Connect boxes to the same thermostat

93. Connect boxes to the same thermostat
Place identity points on the atoms of the ligand

94. Connect boxes to the same thermostat
Place identity points on the atoms of the ligand
Copy those points into the water box to identify a cluster

95. λ"="0.0" λ"="0.3" λ"="0.6" λ"="1.0"
• Binding"free"energy"is"calculated"by"running"
simula:ons"across"λ."Using"one"8>core"node,"one"
free"energy"takes"24>48"hours"to"compute"
• Implemented"in"Sire:"hHp://siremol.org""
• Woods,"J"Chem"Phys,"Vol"134,"p054114,"2011"
• "hHp://dx.doi.org/10.1063/1.3519057"

96. Reflection Sphere
• Only waters
whose centers
are inside the
sphere can move
• Any move that
takes the center
of a water outside
the sphere is
reflected back
into the sphere
• This prevents
waters from
leaving

97. Grid Electrostatics
• Interactions inside
reflection sphere
calculated normally
• Interactions between
reflection sphere atoms
and atoms within buffer
(dotted sphere)
calculated normally
• Coulomb interactions
between reflection
sphere and fixed atoms
outside the buffer are
calculated using a pre-
computed cubic grid

98. Grid Electrostatics
• Use of pre-computed
grid means that there is
no penalty to using a
long-range electrostatic
cutoff
• Compatible with
advanced boundary
conditions, such as
reaction field or force-
shifted cutoff
• Fine grid (0.5 Å) and
tri-linear interpolation
give high accuracy
compared to direct
calculation