M.C.

Childers 2020

engineering the cardiovascular system

with atomic simulations
Long term goals: leveraging molecular models to
engineer cardiovascular therapies
Relevance
- Enhancing myosin activation/relaxation through small molecules
is a viable approach to treat heart failure

- The naturally occurring nucleotide 2’-deoxy-ATP is one such

small molecule

- dATP increases hydrolytic activity of myosin, increases the

magnitude and rate of contraction in demembranated myocytes;
in animal models, and in failing dog & human myocardium

Image Source: AHA, https://www.heart.org/en/news/2019/01/31/cardiovascular-

diseases-affect-nearly-half-of-american-adults-statistics-show
Myosin and dATP

Elevated levels of
dATP enhance myosin
function and improve
cardiac output in
multiple systems
How does dATP exert its effects?
 Part I
A crash course in structural biology
Molecular Architectures – ‘Form follows Function’

The field of structural biology is concerned with

the three dimensional structures of biological
macromolecules, how those structures are
formed, and how those structures relate to
biological functions.

Several techniques are available to determine

protein structures, the most popular are X-ray
crystallography, NMR spectroscopy, and cryo-
electron microscopy.

The Protein Data Bank (PDB, www.rcsb.org) is a

free, open-access database that contains >
156,000 structures.

Novus Biologicals
Molecular Architectures – ‘Form follows Function’

The field of structural biology is concerned with

the three dimensional structures of biological
macromolecules, how those structures are
formed, and how those structures relate to
biological functions.

Several techniques are available to determine

protein structures, the most popular are X-ray
crystallography, NMR spectroscopy, and cryo-
electron microscopy.

The Protein Data Bank (PDB, www.rcsb.org) is a

free, open-access database that contains >
156,000 structures.

RCSB Molecule of the Month – David Goodsell

Molecular Architectures – ‘Form follows Function’

The field of structural biology is concerned with

the three dimensional structures of biological
macromolecules, how those structures are
formed, and how those structures relate to
biological functions.

Several techniques are available to determine

protein structures, the most popular are X-ray
crystallography, NMR spectroscopy, and cryo-
electron microscopy.

The Protein Data Bank (PDB, www.rcsb.org) is a

free, open-access database that contains >
156,000 structures.
'Photographing Proteins’: X-ray Crystallography

MRC Laboratory of Molecular Biology; Nature 185, 416–422 (1960); NASA Marshall Space Flight Center; Thomas
Splettstoesser
Molecular Architectures – ‘Form follows Function’

The field of structural biology is concerned with

the three dimensional structures of biological
macromolecules, how those structures are
formed, and how those structures relate to
biological functions.

Several techniques are available to determine

protein structures, the most popular are X-ray
crystallography, NMR spectroscopy, and cryo-
electron microscopy.

The Protein Data Bank (PDB, www.rcsb.org) is a

free, open-access database that contains >
156,000 structures. These structures can be
visualized with molecular graphics programs
like UCSF Chimera (www.cgl.ucsf.edu/chimera/)
 Part II
Computational methods to study
protein dynamics
 Problem: many approaches yield results averaged over
molecules over time…

12
MRC Laboratory of Molecular Biology; Nature 185, 416–422 (1960)
 … but proteins are dynamic, heterogeneous molecules, and those
dynamics are intricately connected to protein structure and function

13
Curr. Opin. Pharm. 10 (6) 745-752. (2010)
Solution: computational molecular simulations

14
‘Central dogma’ of MD

- Biological activity is the result of time-dependent interactions between

molecules across multiple temporal and spatial scales

- Microscopic (atomic) behavior is related to macroscopic observables

- The time-dependent microscopic behavior of a system can be described by a

mathematical model
Mathematical Model: Classical Mechanics of atomic systems

𝑑𝑣# 𝑑- 𝑟#
−∇# 𝐸 = 𝐹# = 𝑚# ⋅ 𝑎# = 𝑚# =𝑚 -
𝑑𝑡 𝑑𝑡

Three things are required to run an MD

simulation: a set of atomic coordinates for the
system of interest, a force field, and a set of
‘rules’ for running the simulation (a.k.a. the
simulation package)
Obtaining Initial Coordinates
The Force Field
The Force Field
Force field = potential energy function + associated parameters

1 -
1 -
𝐸 𝑅 = 0 𝐾 𝑏 − 𝑏; + 0 𝑘 𝜃 − 𝜃; + 0 𝐾F 1 + cos 𝑛𝜙 − 𝛿
2 9 2 B
12345 =3>?@5 4#D@4E=?5

Q- R
𝑟;,#O 𝑟;,#O 𝑞Q 𝑞-
+ 0 𝜀#O − 2𝜀#O + 0
𝑟#O 𝑟#O 𝐷𝑟
31 M=#E5 31 M=#E5
#,O #,O

Each of the parameters shown is usually determined through

quantum-level calculations or through data obtained from model
small molecules.
The simulation package

A simulation package is a collection of algorithms that specify how the simulation

should be performed, note that this is distinct from the force field, although the two
are frequently conflated.

Some Example Simulation Packages:

AMBER -> Assisted Model Building with Energy Refinement; UCSF

GROMACS ->GROningen Machine for Chemical Simulations; U of Groningen
ilmm -> in lucem molecular mechanics; U of Washington
NAMD -> Nanoscale Molecular Dynamics; U of Illinois at Urbana-Champaign
Putting it all together

Explore molecular mechanisms of protein function, understand structural/dynamical consequences of modifications

(e.g. post-translational modifications, amino acid mutations, ligand binding), create ‘designer’ proteins, design small
molecules to perturb protein dynamics, and much more!
 Part III
dADP modulates post-powerstroke
dynamics in myosin II

© M.C. Childers 2019

Myosin and dATP
Myosin is a promiscuous enzyme that can utilize a variety of
nucleotides as an energy source.

ADP

dADP
Objectives

• What is the significance of 2 atoms? Or, by what mechanisms does dATP

enhance myosin function?

• In the post-powerstroke state, what conformations of myosin are promoted when

dATP is used as an energy source?

• How do changes in myosin:nucleotide interactions affect functional regions in

myosin?
Myosin chemomechanical cycle
MD Simulations of post-powerstroke myosin
Simulations of myosin in complex with either ADP + Mg2+ or dADP + Mg2+ were performed using
in lucem molecular mechanics. Simulations were performed at 310 K in triplicate for 50
nanoseconds.

ADP

dADP
Nucleotide dynamics: distinct flexibilities of dADP
and ADP
Dynamic changes in the binding pocket are
transmitted to functional sites
Dynamic changes in the binding pocket are
transmitted to functional sites
Connecting perturbations and responses

Represent myosin as a network of interactions Identify interactions that differ in the ADP and dADP simulations
Connecting perturbations and responses
Connecting perturbations and responses
Connecting perturbations and responses
 Part IV
“Scallops are expensive, so they
should be treated with some class”
– Jeff Smith
Model preparation: Mapping the chemomechanical cycle

1QVI 2OTG
(pre-powerstroke, ADP + Pi) (post-powerstroke / ADP)

1KK8 1SR6
(post-rigor, ATP) (near-rigor / Nucleotide Free)
1QVI Simulation Prep
Final Model Selection The terminal end of the myosin tail along with the essential light chain were truncated
from the structure. Recall, the CPU effort required to run a simulation scales exponentially with the number
of atoms. Few Atoms = Faster Simulations

Chain A (myosin heavy chain) residues 1 – 810 + ADP + Pi + Mg2+

Chain Z (essential light chain) residues 1 – 156 + Mg2+

Old (1VOM, D. discoideum) simulations = heavy chain residues 1 – 762 + Pi + Mg2+

1QVI Simulation Prep
New: Geometry = Truncated Octahedron; Volume = ~2,450,000 Å3, Num. Atom = ~240,000
Old: Geometry = Rectangular Hexahedron; Volume = ~900,000 Å3; Num. Atoms = ~86,000
Again we find that dADP is less stable in the binding
pocket
Dynamics of actin binding residues

dADP increased the solvent exposure of actin binding residues in myosin

Dynamics of actin binding residues
Thank You!
HAMM Lab Daggett Group
• Mike Regnier • Valerie Daggett
• Sarah Nowakowski • Clare-Louise Towse
• Gene Hopping
Funding Sources This work was supported by the Bioengineering Cardiovascular Training Grant (NIH/NIBIB
T32EB1650 to M.C.C.) and the National Institutes of Health (GMS R01 95808, GM 50789 to V.D.).

We thank the National Energy Research Scientific Computing Center, supported by the DOE Office of
Biological Research, which is supported by the US Department of Energy under contract number DE-AC02-
05CH11231, for providing computing time. This work used the Extreme Science and Engineering Discovery
Environment (XSEDE) resource STAMPEDE through allocation MCB160012. XSEDE is supported by
National Science Foundation grant number ACI-1548562.

© M.C. Childers 2019

Connection with experiment

Nowakowski et al. 2017 Protein Science 26 (4)

Dynamic changes in the binding pocket are
transmitted to functional sites
ADP and dADP form distinct interactions with myosin

ADP (0 ns) ADP (50 ns) dADP (50 ns)

Nucleotide dynamics: distinct flexibilities of dADP
and ADP
ADP and dADP form distinct interactions with myosin