Transcript PPT - Center for Computation & Technology

Program No.: 842.1
Abstract No.: 2468
Dynamics differences of SAM-I riboswitch aptamer between SAM bound
and without SAM: insight into conformational rearrangement
Wei Huang1, Fareed Aboul-ela1, Joohyun Kim2, Shantenu Jha2
1. Department of Biological Science, 2. Center for Computation & Technology, Louisiana State University, Baton Rouge, Louisiana 70803
Introduction
A riboswitch is an autonomous cis-regulatory element that
controls gene expression in response to the signals from small
molecule metabolites, such as amino acids, nucleotides, or enzyme
cofactors etc [1]. The SAM-I riboswitch is one member of this
regulatory family that can control the expression of biosynthetic
proteins involved in sulfur metabolism through conformational
change upon the binding of S-adenosylmethionine (SAM). In the
presence of SAM, pairing non-adjacent regions forms the P1 helix
of the anti-antiterminator (AAT) structure, which prevent
formation of antiterminator (AT). Meanwhile, this facilitates
formation of the rho-independent terminator (T) hairpin to achieve
transcriptional attenuation [2].
Principal Component Analysis
RMSD of Binding Pocket
The MD simulation samples the 3N-dimensional space
corresponding to the positions of the N atoms. We used PCA to extract
the essential dynamics from the trajectories. These essential dynamics
are anharmonic motions, which are assumed to be the motions relevant
for the functions of biomolecules [4]. The approach therefore contrasts
with conventional normal mode dynamics, which detects relatively
trivial harmonic motions. Root-mean-square-fluctuation (RMSF) of
SAM_TRAJ and WoSAM_TRAJ along their first 5 eigenmodes on an
atomic basis are displayed (color scheme is the same as the secondary
structure representation). In both trajectories, the P4 helix is the most
flexible region. This is due to the P4 helix has few tertiary contacts
with the other part of the RNA. Residues showing spike patterns are
highlighted. Residues (A14, U34 and A51) that have spike pattern in
both trajectories are highly exposed to water solvent in the
crystallographic structure (PDB: 2GIS) [3]. These residues only show
flexibility in WoSAM_TRAJ are involved in formation of the binding
pocket (A10, A46) and the pseudoknot (G68). The jump of residue A9
observed in SAM_TRAJ is in the junction J1/2.
Non-adjacent Dinucleotide Stack
Although the structure of SAM-I riboswitch aptamer domain
has been solved via X-ray crystallography [3], it is just a static view
of how SAM binds to SAM-I riboswitch. In this study, molecular
dynamics (MD) simulations are performed on SAM-I riboswitch
aptamer with SAM and without SAM for up to 200 ns. Principle
component analysis (PCA) is applied to explore the global dynamics
differences of SAM-I riboswitch aptamer between SAM present and
SAM absent. MD simulation results provide us insights into the
conformational changes observed in the SAM-I riboswitch.
Distance monitor and sample snapshots of
the A9 and U63 non-adjacent stack in SAM_TRAJ
(red) and WoSAM_TRAJ (blue). The distance
between atom N6 on the nucleotide A9 and atom
O4 on the nucleotide U63 was used to monitor this
non-adjacent dinucleotide stack. Distances around
5 Å indicate stacked state, while distances much
larger than 5 Å are interpreted as unstacked. Some
.
sample
snapshots are presented. Multiple instances
of the transient stack take place, typically holding
for a period of tens of nanoseconds.
Molecular Dynamics Simulation
The aptamer domains of T.tengcongensis SAM-I riboswitch
and its complex with SAM has been submitted to MD simulation for
200 ns using an explicit water solvent model. The 2.9 Å crystal
structure of SAM bound to the aptamer domain of SAM-I
riboswitch [2] is used as the starting point.
Coordination of the Specific Binding Magnesium
The residues that have atoms within 5 Å of SAM were defined
as the binding pocket (6-8, 10-12, 44-47, 57-59, 87-89). Time
evolution of root-mean-square-deviation (RMSD) of the binding
pocket and the ligand SAM. In the trajectory simulated in the
presence of SAM (SAM_TRAJ, red), the RMSD is stable, while in
the absence of SAM (WoSAM_TRAJ, blue), the RMSD increases
until approximately 100 ns.
Conclusions and Future Work
PCA combined with the time series of RMSD clearly suggest
that a core region of the aptamer structure, defined as those
secondary structure elements that contact SAM (P1, J1/2 and P3)
are stabilized when SAM is bound. P4 and some regions of P2,
which do not have direct contact with SAM, undergo more dynamic
fluctuations in the SAM_TRAJ trajectory.
In addition, the monitor of magnesium coordination suggests
that J1/2 cannot maintain its compact configuration as seen in the
crystallographic structure in the absence of SAM However, the
magnesium coordination is preserved throughout the simulation in
the presence of SAM. This result suggests that the SAM binding
affects the placement of J1/2.
The coordination of the specific binding magnesium and
phosphate atom on A9 is coupled with this dinucleotide stack state.
On the one hand, when the stack is lost, the coordination of the
magnesium and phosphate atom on A9 is shortened. On the other
hand, when the distance between the magnesium and phosphate
atom on A9 increases by a small but significant extent, formation of
the stack appears. It is possible that this non-adjacent dinucleotide
stack stabilizes the tertiary interactions between J1/2 and J3/4.
In the future, isotropic reorientational eigenmode dynamics
(iRED) analysis [5] will be applied on both trajectories to extract
the information about the dynamical correlation. In addition,
biochemical and biophysical experiments will be carried out to
validate the results of our simulations.
Reference
PDB ID: 2GIS
The simulations with SAM are designated as SAM_TRAJ, while the
simulations without ligand are denoted as WoSAM_TRAJ. The
overall deviation of coordinates from the starting structure and
from an average structure are explored under the guidance of PCA.
In this way, we could determine when our simulations had
equilibrated, and detect signs of instabilities in coordinates which
might indicate intrinsic problems with the structure.
The magnesium ion apparently counters the electrostatic repulsion between the phosphate backbone of J1/2
and that of J3/4. Additionally, this magnesium binding site is close to the SAM binding pocket. We monitored the
distances of this magnesium to negatively charged atoms on the RNA during our simulated trajectories . In
WoSAM_TRAJ, the contact distances between the magnesium ion and the phosphate backbone of J1/2 lengthen,
while those with the phosphate backbone of J3/4 hold constant. The S-turn motif in J3/4 is preserved during the
simulation. In SAM_TRAJ, all of the magnesium distances are constant through the simulation. This result
suggests that binding of magnesium and SAM might have a synergistic effect on the stabilization of the J1/2
junction.
[1] Mandal, M. et al. (2004). Nat Rev Mol Cell Biol, 5, 451 - 463.
[2] Brooke, A.M.M. et al. (2003) Proceedings of the National Academy of
Sciences of the United States of America, 100, 3083-3088.
[3] Montange, R.K. et al. (2006). Nature, 441, 1172-1175.
[4] Amadei, A., Linssen, A.B. and Berendsen, H.J. (1993) Essential dynamics
of proteins. Proteins, 17, 412-425.
[5] Prompers, J.J. et al. (2002) J Am Chem Soc, 124, 4522-4534.
Acknowledge
This work is supported by a Faculty Research Grant from the LSU
Office of Research and Economic Development. We thank other members in
Dr. Aboul-ela’s lab for useful discussions.We thank the LSU & LONI HPC
support for their help during the use of High Performance Computing
machines.