Insights into the Ligand Specificity of

Guanidinium III Riboswitch

with the help of Molecular Dynamics
Simulations

SUPERVISOR PRESENTED BY

Dr. Purshotam Sharma Amanpreet Singh

 Riboswitches

• Structural element in mRNA.

• Located in the 5ʹ untranslated region of mRNA.
• Couples with small-molecules to bring conformational
changes.
• Regulates gene expression.
• Binding of ligand is associated with metabolic role of gene
product.
• Very sensitive and specific to ligand binding.

Battaglia et al. RNA Biol. 2017, 578-585.

Huang et al. Cell Chem. Bio. 2017, 24, 6, 695-702.
 Riboswitch structure and mechanism
Structure has two Domains:
• Aptamer Domain – ligand binding site (highly conserved).
• Expression Platform – brings conformational change to control gene
expression in response to ligand binding (less conserved).

Sherlock et al. Biochemistry 2017, 56, 2, 352-358.

Sherlock et al. Biochemistry 2017, 56, 2, 359-363.
Guanidine-Sensing class of Riboswitches

Breaker et al. Mol. Biochemistry 2017, 56, 2, 345-347.

Huang et al. Cell Chem. Biol. 2017, 24, 6, 695-702.
Huang et al. Cell Chem. Biol. 2017, 24, 11, 1407-1415.
 Aptamer Domain of the Guanidine III riboswitch

Structure Sequence
(PDB Code:5NWQ)

L1

Cartoon Representation Sequence Representation

Cognate Ligands
 Methodology
• Molecular Dynamics Simulations were performed using
Amber 14 using ff99bsc0χOL3 force field.
 Methodology
• Energy minimization in two steps.
• Equilibration step involving gradual heating.
• Production simulation of 100 ns each.
Ligand Time Repetition
Open state (without 100 ns 5 Times
ligand)
Guanidinium (Gnm) 100 ns 5 Times

Arginine (Arg) 100 ns 5 Times

Urea 100 ns 5 Times

Siladitya et al. J. Mol. Graph. Model. 2019, 88, 282-291.

Battaglia et al. Wiley Interdiscip. Rev. RNA 2018, 9, 5, e1482
 Root Mean Square Deviations (RMSD)
G-III state Open State Gua Bound Arg Bound State Urea Bound
State State
RMSD (Å) 0.720 (1.018) 0.802 (1.135) 0.664 (0.939) 1.012 (1.432)
Avg (Stdev.)
Root Mean Square Fluctuation (RMSF)
P1 L1 P2 L2 P1 L2 P2
Root Mean Square Fluctuation (RMSF)
P1 L1 P2 L2 P1 L2 P2
Superposition of the Snapshots of the G-III
Riboswitch Aptamer Simulations

L2 L2 L2 L2
Dynamic Cross Correlation Maps
 Interaction Energies
Type of Residue number Gnm bound Arg bound state Urea bound
interaction state state
energies
Hydrogen bonding G7---(ligand)42 -38.6 (4.3) -38.9 (2.2) -1.7 (3.7)
energy A8---(ligand)42 -3.0 (3.0) -11.7 (4.0) -0.8 (1.8)
(kcal/mol)
G17---(ligand)42 -38.6 (4.8) -36.6 (2.3) -0.8 (3.4)
Stacking energy C6---(ligand)42 -10.2 (5.6) -1.0 (1.2) -3.2 (4.5)
(kcal/mol)

C6

G17
G7

A8
 Conclusion
• The triple helix formation (involving L1 and P2) is stabilized by ligand
binding – decrease in RMSF value.
• Urea – less stabilized compared to Arg and Gnm due to lack of
formation of hydrogen bonds at the binding site.
• Arginine and urea less stabilized via π - cation stacking interaction
compared to Guanidinium ion.
• Correlation in motion – on ligand binding.

Future Work
• To analyze the essential dynamics of aptamer domain Principal
Component Analysis will be performed.
• In order to study conformational changes at different regions a
complete analysis of dihedral angles will be done.
• Various structural parameters will be analyzed to understand rotational
and translational relationships involved.