Molecular Dynamics: A Tool to Understand Ligand-Receptor
Interactions
F. Javier Luque
Department of Nutrition, Food Sciences, and
Gastronomy
Institut de Biomedicina
University of Barcelona
Text mining and chemoinformatics sketch a
comprehensive picture of the interference of dietary
components
with
pharmacokinetics
and
pharmacodynamics processes of drugs
Bioactive phytochemicals from food source and biological target in colon
cancer
Plos Comput Biol 2014, 10, e1003432; 2015, 10, e1004048
How does a small molecule exert its biological effect?
Tripsin-benzamidine complex
500 trajectories (100 ns each)
187 produced binding events
RMSD < 2 Å compared to the crystal
structure
Predicted binding free energy: -5.2
kcal/mol
(experimental value: -6.2 kcal/mol)
Binding pathway involves two metastable
intermediate states
Proc Natl Acad Sci USA 2011, 108, 10184.
Molecular dynamics
Non bondedterms
Bondedterms
Other
Polarization
E  Estr  Ebnd  Etor  Enb  Eother
E str 
K
str
(l  l o ) 2
ang
(   o ) 2
bonds
Ebnd 
K
angles
3
Vn
( 1  cos n   )
2
n 1
Etor  
tor
Restraints
Qm Qn
m , n  ( Rmn ) Rmn
Eele  
 Aij Cij 
Evw    12  6 

Rij 
i , j  Rij
Characteristic time scales for protein motions
Bovine
pancreatic
trypsin inhibitor
(BPTI)
Chicken villin
Headpiece
subdomain
Protein folding
100 μs - 1 ms
1 μs
9.2 ps
MacCammon, Gelin,
Karplus,
Nature, 1977, 267, 585
Duan, Kollman,
Science, 19798, 282, 740
Lindorff-Larsen, Piana, Dror,
Shaw,
Science, 2011, 334, 517
Molecular dynamics
•
Information on the time evolution of conformations of (bio)molecular systems
•
Properties of the system: structure, dynamics, kinetics, thermodynamics, mechanisms
•
Relationships between structure, dynamics and function in biomolecules
7
Mechanisms of ligand activity: allosteric regulation
Inhibition of GSK3-beta by palinurin
Mechanisms of ligand activity: alloenzyme activator
Activation of AMPK by A-769662
Sensitivity of biological activity to mutations
Role of distant mutants in androgen receptor
Binding/Unbinding of small molecules by carriers
Retinol transport by CRBP-I and CRBP-II
Mechanisms of ligand activity: allosteric regulation
Inhibition of Glycogen Synthase Kinase 3 by Palinurin
• In Alzheimer’s disease, it has been
linked to tau hyperphosphorylation,
amyloid deposition, and neuronal
death.
• Allosteric modulation offers a
subtle, selective regulation, and
minimize unwanted effects due to
competition at the ATP binding site.
• Two orphan sites: 5 and 6
VP0.7
Palomo V., et al., J. Med. Chem., 2011, 54,
8461
Palinurin
1.010 6
5.010 5
-0.2
Ircinia dendroides
0.2
0.4
4.010 5
2.010 5
-0.2
0.2
1/[ATP] (M)-1
-5.010 5
Ircinin
6.010 5
1/V0 (M/30min)-1
1/V0 (M/30min)-1
1.510 6
Ascorbic acid
derivatives
Asc1
Ircinin-1
Asc2
Ircinin-2
Asc3
0.4
1/[GS2] (M)-1
-2.010 5
GSK-3b inhibition by palinurin can not be competed
out by ATP nor peptide substrate
The binding site for the inhibitor lays outside the
binding pockets for both ATP and the substrate.
Palinurin binds to the pocket site 5
Palinurin binding site
(pocket 5)
Substrate binding site
ATP binding site
Kinase selectivity
CK2
IC50 (mM)
pIC50
GSK-3b
1.9
5.72
GSK-3a
1.6
5.79
CDK5
> 25
< 4.6
CDK1
> 100
< 4.0
MAPK
> 100
< 4.0
CK2
> 100
< 4.0
CDK1
CDK5
Palinurin binding alters the accessibility of the γ-phosphate of ATP.
Palinurin binding affects the flexibility glycine-rich loop
(via the interaction between Ser66 at the tip of the loop and the –phosphate of
ATP)
Holo
complex
Glycine-rich loop
Holo complex
+
palinurin
Mechanisms of ligand activity: enzyme activator
AMPK (AMP-activated protein kinase)
•
•
•
Ser/Thr protein kinase
Activated by low levels of ATP and high levels of
AMP/ADP
Sensor of energy homeostasis in the cell
LKB1
CaMKKb
Upstream Kinases
AMPK
Catabolic Pathways
activated by the
activated AMPK
Mitochondrial
biogenesis oxidative
metabolism PGC-1a? /
SIRT1?
Downstream Kinases
Glycolysis
PFKFB2/3
Autophagy
ULK1
Nat Cell Biol. 2012, 13, 1016. BMC Biology 2013, 11, 36.
Anabolic Pathways
inhibited by the
activated AMPK
Transcription of
ribosomal RNA
TIF-1A
Translation of
ribosomal proteins
mTORC1*
Synthesis of
fatty acids
ACC1
Protein
synthesis
mTORC1*
13
Structure vs. Function
b
a 
Dephosphorylation
@Ser108
Phosphorylation
@Thr172
≈1000x
b
a 
>90x
A-769662
AMP
2- 4x
2-13x
b
a 
2-13x
Dephosphorylation
@Thr172
Autophosphorylation
@Ser108
b
a 
Chem. Biol., 2014, 21, 619.
14
How does A-769662 trigger AMPK activation?
APO
HOLO
HOLO + ATP
15
CBM
C-interacting
helix
P-loop
αC-helix
Active
loop
16
The effect of activator and ATP in the conformational and dynamic behaviour
17
The activator acts like a glue between the α-kinase domain
and the CBM domain of β-subunit
18
Allosteric Hypothesis
The activator pre-configurates the ATP-Binding Site
19
Distant mutant selection in androgen receptor
•
Drug target against metastatic prostate cancer
•
Ligand-binding domain (LBD) is the site of hormone
binding and co-regulatory protein interactions (via
AF2 surface, involving residues from H3, H4, H12)
•
The impact of selective mutations is not completely
understood.
•
V757A, H874Y and Q798E are remotely located
from the ligand pocket, and exhibit different
degrees of simultaneous gain/loss of function.
•
Understanding their effect on protein activity may
explain the allosteric behavior of AR
Wild-type AR response to different ligands
Comparison of mutant and WT-type AR response to ligands
Each mutation has a unique character, with outcomes largely dependent on the concentration and
chemical nature of the ligand.
However, no apparent structural relationship appear between the mutated residues and the possible
activation/inactivation effect.
V757A
Simulation sytems
Q798E
H874Y
Conformational selection model in AR
Relative mobility of residues in the MD simulations
(wider loops and warmer colours indicate increased mobility; the ellipse marks the position of the AF2
site)
A - apo
B - DHT
H1
H4
H4
H3
H3
H3
b1
b2
H12
Representation of the conformational ensemble
(ARAPO: orange, AR-DHT: yellow; AR-DHT-SRC: red)
D
H1
H1
H4
H12
C - SRC
b1
b2
H12
b1
b2
Pairwise residue interaction energy – Principal Component Analysis
Allosteric transmission does not occur through a single pathway; rather it involves throughout the
protein, affecting some more areas than others (i.e., C-terminal tail, H3, H9, H5).
It also hints at possible communication pathways
Due to its high connectivity, central location and tight binding with the protein, the hormone (DHT) acts as
a linking hub, connecting different functional parts, such as AF2 (binding of co-regulators) and the H5S1 loop (presumably involved in AR dimerization)
Retinol transport by CRBP-I and CRBP-II
Retinoids are essential for many physiological processes
(cell growth and differentiation, morphogenesis and vision)
Highly insoluble: they have to be transported by specific proteins
Cellular Retinol Binding-Proteins (CRBPs)
25
N MR
X -ray
apoCRBP-I
holoCRBP-I
apoCRBP-II
PDB ID 1JBH
PDB ID 1KGL
PDB ID 2RCQ
+
B-factor
holoCRBP-II
PDB ID 2RCT
–
Lipocalin fold
Sequence homology close to 70%
Binding affinity of retinol to isoforms I and II leads to dissociation constants (Kd) of
0.1 nM for CRBP-I and of 10 nM for CRBP-II.
NMR H/D exchange experiments suggest that the distinct affinity might arise from
differences in conformational flexibility
J. Biol. Chem. 1991, 266, 3622; J. Biol. Chem. 2002, 277, 21983..
26
Essential Dynamics Analysis vs. NMR Proton Exchange
apoCRBP-I
holoCRBP-I
apoCRBP-II
holoCRBP-II
+ Mobile regions
- Mobile regions
Binding of retinol promotes the stiffness of both CRBP isoforms, I and II
The portal site appears to have a different flexibility in both isoforms.
Is this the limiting step for retinol binding?
J. Lip. Res. 2010, 51, 1332.
27
Enhanced sampling: Opening of the Entry Portal-Site
28
Enhanced sampling: Binding/Unbinding
29
CRBP - I
CRBP – II
-5.5 ± 0.5
-6.2 ± 0.5
ApoOPEN
+6.4 ± 0.5
HoloOPEN
-12.3 ± 0.5
-11.4 Kcal/mol
ApoCLOSE
HoloCLOSE
ApoOPEN
HoloOPEN
+7.5 ± 0.5
-6.1 ± 0.5
-4.8 Kcal/mol
ApoCLOSE
EX PERIMEN TAL vs.T H EORET ICAL RESULT S
HoloCLOSE
-5
6.0
Kd (CRBP-I) = koff / kon = 0.1 nM ≅ DG = -13.0 Kcal/mol
-7
Kd (CRBP-II) = koff / kon = 10 nM ≅ DG = -9.5 Kcal/mol
-9
-9.5
- 11
- 13
-12.0
-13.0
33
30
31