Name: structure-based simulation and sampling of transcription factor protein movements along dna from atomic-scale stepping to coarse-grained diffusion
Uploaded: 2022-03-01T13:10:00
Description: Watch this Scientific Journal Video about Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion at JoVE.com

This work has been supported by NSFC Grant #11775016 and #11635002. JY has been supported by the CMCF of UCI via NSF DMS 1763272 and the Simons Foundation grant #594598 and start-up fund from UCI. LTD has been supported by Natural Science Foundation of Shanghai #20ZR1425400 and #21JC1403100. We also acknowledge the computational support from the Beijing Computational Science Research Center (CSRC).

1. Construction of the Markov state model (MSM) from atomic MD simulations
<ol>
	<li>Spontaneous protein stepping pathway and initial structures collection
	<ol>
		<li>Use a previously obtained 10-&#181;s all-atom MD&#160;trajectory8 to extract 10000 frames evenly from a &#34;forward&#34; 1-bp stepping path (i.e., one frame for each nanosecond). The total number of frames needs to be sufficiently large to include all representative conformations.</li>
		<li>Prepare the transitio...

This work addresses how to conduct structure-based computational simulation and samplings to reveal a transcription factor or TF protein moving along DNA, not only at atomic detail of stepping, but also in the processive diffusion, which is essential for the facilitated diffusion of TF in the DNA target search. To do that, the Markov state model or MSM of a small TF domain protein WRKY stepping for 1-bp along homogeneous poly-A DNA was first constructed, so that an ensemble of protein conformations on the DNA along with ...

Transcription factors (TF) search for the target DNA to bind and regulate gene transcription and related activities1. Aside from the three-dimensional (3D) diffusion, the facilitated diffusion of TF has been suggested to be essential for target DNA search, in which the proteins can also slide or hop along one-dimensional (1D) DNA, or jump with intersegmental transfer on the DNA2,3,4,5,6,7.
In a recent study, we have conducted tens of microseconds (&#181;s) all-atom equilibrium molecular dynamics (MD) simulations on a plant TF - the WRKY domain protein on the DNA8. A complete 1-bp stepping of WRKY on poly-A DNA within microseconds has been captured. The movements of the protein along the DNA groove and hydrogen bonds (HBs) breaking-reforming dynamics have been observed. While such a trajectory represents one sampled path, an overall protein stepping landscape is still lack of. Here, we show how to expand computational samplings around the initially captured protein stepping path with the constructed Markov state model (MSM), which have been implemented widely for simulating a variety of biomolecular systems involving substantial conformational changes and time-scale separation9,10,11,12,13,14,15,16,17,18,19. The purpose is to reveal the conformational ensemble and meta-stable states of the TF protein diffusion along DNA for one cyclic step.
While the above MD simulation reveals atomic resolution of the protein movements for 1 bp on the DNA, the structural dynamics of long-time processive diffusion of the TF along DNA at the same high-resolution is hardly accessible. Conducting coarse-grained (CG) MD simulations at residue level is however technically approachable. The CG simulation time scale can be effectively extended to tens or hundreds of times longer than the atomic simulations20,21,22,23,24,25,26,27,28,29. Here, we show the CG simulations conducted by implementing the CafeMol software developed by Takada lab30.
In current protocol, we present the atomic simulations of the WRKY domain protein along poly-A DNA and the MSM construction first, which focus on sampling the protein stepping motions for only 1 bp along DNA. Then we present the CG modeling and simulations of the same protein-DNA system, which extend the computational sampling to the protein processive diffusion over tens of bps along DNA.
Here, we use GROMACS31,32,33 software to conduct MD simulations and MSMbuilder34 to construct the MSM for sampled conformational snapshots, as well as to use VMD35 to visualize the biomolecules. The protocol requires that the user to be able to install and implement the software above. The installation and implementation of the CafeMol30 software is then necessary for conducting the CG MD simulations. Further analyses of the trajectories and visualization are also conducted in VMD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CafeMol</td>
			<td>Kyoto University</td>
			<td></td>
			<td>coarse-grained (CG) simulations</td>
		</tr>
		<tr>
			<td>GROMACS</td>
			<td>University of Groningen Royal Institute of Technology Uppsala University</td>
			<td></td>
			<td>molecular dynamics simulations software</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>Numerical calculation software</td>
		</tr>
		<tr>
			<td>MSMbuilder</td>
			<td>Stanford University</td>
			<td></td>
			<td>build MSM</td>
		</tr>
		<tr>
			<td>VMD</td>
			<td>UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN</td>
			<td></td>
			<td>molecular visualization program</td>
		</tr>
	</tbody>
</table>

structure-based simulation and sampling of transcription factor protein movements along dna from atomic-scale stepping to coarse-grained diffusion

One-dimensional (1-D) sliding of transcription factor (TF) protein along DNA is essential for facilitated diffusion of the TF to locate target DNA site for genetic regulation. Detecting base-pair (bp) resolution of the TF sliding or stepping on the DNA is still experimentally challenging. We have recently performed all-atom molecular dynamics (MD) simulations capturing spontaneous 1-bp stepping of a small WRKY domain TF protein along DNA. Based on the 10 &#181;s WRKY stepping path obtained from such simulations, the protocol here shows how to conduct more extensive conformational samplings of the TF-DNA systems, by constructing the Markov state model (MSM) for the 1-bp protein stepping, with various numbers of micro- and macro-states tested for the MSM construction. In order to examine processive 1-D diffusional search of the TF protein along DNA with structural basis, the protocol further shows how to conduct coarse-grained (CG) MD simulations to sample long-time scale dynamics of the system. Such CG modeling and simulations are particularly useful to reveal the protein-DNA electrostatic impacts on the processive diffusional motions of the TF protein above tens of microseconds, in comparison with sub-microseconds to microseconds protein stepping motions revealed from the all-atom simulations.

Rotation-coupled sliding or 1 bp stepping of WRKY from the MSM construction 
All protein conformations on the DNA are mapped to the longitudinal movement X and rotation angle of the protein COM along DNA&#160;(see Figure 3A). The linear coupling of these two degrees indicates rotation-coupled stepping of the WRKY domain protein on the DNA. The conformations can be further clustered into 3 macrostates (S1, S2, and S3) in the MSM. The forward stepping of WRKY then follows...

Watch this Scientific Journal Video about Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion at JoVE.com

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion

The goal of this protocol is to reveal structural dynamics of one-dimensional diffusion of protein along DNA, using a plant transcription factor WRKY domain protein as an exemplary system. To do this, both atomistic and coarse-grained molecular dynamics simulations along with extensive computational samplings have been implemented.

One-dimensional (1-D) sliding of transcription factor (TF) protein along DNA is essential for facilitated diffusion of the TF to locate target DNA site ...

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