Revealing Atomic-scale Molecular Diffusion of a Plant Transcription Factor WRKY domain protein along DNA

Abstract

designed the study. Liqiang Dai conducted the MD simulations and coarse-grained simulations and analyzed the related data. Yongping Xu solved the crystal structure of WRKY-DNA complex and conducted ITC experiment. Zhenwei Du conducted the single-molecule fluorescence experiments of WRKY diffusing on DNA. AbstractTranscription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here we constructed a TF model system of the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on the DNA employing all-atom molecular dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to the Crick strand of DNA with significantly stronger energetic association than to the Watson strand. The preferential binding becomes highly prominent from non-specific to specific DNA binding, but less distinct from static binding to diffusive movements of WRKY on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base pair (bp) stepping cycle of WRKY tracking along major groove of DNA with homogenous (AT)n sequence, as individual protein-DNA contacts break and reform at the binding interface. Continuous tracking of WRKY forward or backward, with occasional sliding as well as strand crossing to the minor groove of DNA, have also been captured in the simulation. The processive diffusion of WRKY had been confirmed by accompanied single-molecule fluorescence assays and coarsegrained (CG) structural simulations. The study thus provides unprecedented structural dynamics details on the TF diffusion, suggests how TF possibly approaches to gene target, and supports further high-precision experimental follow-up. The stochastic movements revealed in the TF diffusion also provide general clues on how other nucleic acid walkers step and slide along DNA. Significance StatementHow transcription factors search for target genes impact on how quickly and accurately the genes are transcribed and expressed. To locate target sufficiently fast, 1D diffusion of the protein along DNA appears essential. Experimentally, it remains challenging to determine diffusional steps of protein on DNA. Here, we report all-atom equilibrium simulations of a WRKY protein binding and diffusing on DNA, revealing structural dynamics details which have not been identified previously. We unprecedently demonstrate a complete stepping cycle of the protein for one base pair on DNA within microseconds, along with stochastic stepping or sliding, directional switching, and strand crossing. Additionally, we have found preferential DNA strand association of WRKY. These suggest how protein factors approach toward target DNA sequences.

Insights into protein–DNA interactions from hydrogen bond energy‐based comparative protein–ligand analyses

“…For example, most of the DNA binding proteins are transcription factors, which bind to conserved DNA binding sequences while allowing variations at certain sites to regulate gene expression. Recent structural and dynamic analyses have shown that transcription factors typically bind to a preferred strand of the DNA double helix 19,54 . A fine balance of strong and weak HBs helps transcription factors bind to conserved yet different sequences by allowing easier association and disengagement.…”

“…In PD complexes, for example, HBs play a key role in DNA base readout by proteins and act as the major contributor to binding specificity that is vital for the biomolecular function of protein–DNA complexes 15 . The recognition of DNA by proteins is guided by an innate hydrogen‐bonding pattern that generates an initial unstable nonspecific, intermediate complex with high energy 16–19 . While most of this recognition is expected to occur through the signature hydrogen‐bonding pattern in the major groove, many DNA binding proteins also bind to the minor groove through hydrogen bonding and shape readout 15,20 .…”

“…15 The recognition of DNA by proteins is guided by an innate hydrogenbonding pattern that generates an initial unstable nonspecific, intermediate complex with high energy. [16][17][18][19] While most of this recognition is expected to occur through the signature hydrogen-bonding pattern in the major groove, many DNA binding proteins also bind to the minor groove through hydrogen bonding and shape readout. 15,20 Later, this complex transitions to a stable and highly specific low energy state through reversible structural deformations that are also guided by a specific HB pattern.…”

Spontaneous binding of single-stranded RNAs to RRM proteins visualised by unbiased atomistic simulations with rescaled RNA force field

“…The essence of the sliding mechanism employed by HuR RRM3, i.e., local disruption followed by quick global disruption followed by the register shift and restoration, resembles an earlier described translocation mechanism of the WRKY domain protein along DNA helix. (79) We suggest it could be a general strategy employed by proteins specialized on efficient scanning of long nucleic acid sequences without external driving force. In case of HuR RRM3, it would allow it to rapidly search for the RNA sequence whose binding corresponds to thermodynamic minimum while efficiently screening against utterly incompatible sequences (such as the poly-C RNA, see the Supplementary data) by sequestering them in the non-specific and relatively weak pre-binding state.…”

Deprotonation Dynamics of Guanine Radical Cations†

“…A first step toward this direction was made in a recent quantum chemical study: it was shown that a cluster of four water molecules must be taken into account to correctly reproduce the temperature dependence of the reaction rates (G + ) Á → (G-H1) Á in dG determined experimentally as a function of temperature (10). Regarding DNA multimers, theoretical studies should encompass, in addition, molecular dynamics simulations on tens of microseconds, already used to describe interaction of DNA with other biomolecules (63,64). In this way, it should be possible to establish a correlation between DNA topology and (G + ) Á dynamics.…”