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

Summary

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.

Abstract

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 µs 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.

Introduction

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 DNA^2,3,4,5,6,7.

In a recen....

Protocol

1. Construction of the Markov state model (MSM) from atomic MD simulations

1. Spontaneous protein stepping pathway and initial structures collection
   1. Use a previously obtained 10-µs all-atom MD trajectory⁸ to extract 10000 frames evenly from a "forward" 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.
   2. Prepare the transition path with 10000 frames in VMD by clicking File > Save coordinates, type protein or nucleic in selected atoms box and choose fra....

Results

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 (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.......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
CafeMol	Kyoto University		coarse-grained (CG) simulations
GROMACS	University of Groningen Royal Institute of Technology Uppsala University		molecular dynamics simulations software
Matlab	MathWorks		Numerical calculation software
MSMbuilder	Stanford University		build MSM
VMD	UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN		molecular visualization program